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Leica EM AFSRecipe Book

Table of Contents page Introduction 1 List of abbreviations 2 Disclaimer 2 Rules of thumb 3 How to read the protocols 3 Sample Protocols 6 LTE for TEM 6 Various Tissues and Cells 6 Plant Tissue 7 Animal/Human Tissue 31 Viruses, Bacteria, Cells 56 PLT for TEM 66 X-ray microanalysis 71 SEM and LM 74 References 81 All micrographs on title page by Werner Graber, Anatomisches Institut der Universiät Bern, CH

Introduction

1

Introduction In the previous years our PM Team had to face an increasing number of inquiries for freeze substitution protocols. Questions as: Is this medium suitable for my tissue? How long do I substitute? At what temperature? Which resin should I use? are frequently asked. Fortunately, answering is easy: in combination with the Leica EM AFS it takes only a few “rules of thumb” to obtain good results with almost all kinds of specimens. As THE method to prepare cryofixed specimens for ultramicrotomy FS is now more than 40 years of age (Fernández-Morán 1957). The method has been improved and modified ever since (e.g. van Harreveld & Crowelll 1964 for TEM, Barlow & Sleigh 1979 for SEM) and for the absolute beginner the astonishing diversity of protocols, and recipes seems to be a never ending story. This confusing variety is often believed to be “a part of the business”. For example Nicolas & Bassot 1993 state: “In conclusion, the FS protocol must always be adapted empirically. It depends not only on the FS solvent, duration, and ranges of temperature, but also on the nature, size and hydratation of the specimen and on the quality of the FFF [fast-freeze fixation].” Additionally many authors, especially when using “home made” FS units tend to be inaccurate in their “Materials and Methods” section when it comes to cooling rates and polymerisation protocols. The aim of this AFS Recipe Book is to avoid this confusion by giving the reader detailed protocols as a starting point. We are presenting possible methods of dehydration, infiltration and embedding of different types of tissue and microscopic methods. From here on it is up to you to achieve your own routine way for your preferred sample preparation. The Leica EM AFS is a very convenient instrument to perform FS in an uncomplicated, inexpensive and safe way, leading to perfect results. The protocols presented herein are easily adapted to the program steps (T1, S1, T2, …) of the AFS and thus should free up your valuable time for more sophisticated techniques. We are thankful to the many scientist all over the world who contributed to the collection of protocols and we would like to invite you to send in your protocols, micrographs, hints and comments for inclusion in the next edition. We are looking forward to your experiences! Vienna, January 2001 The Leica EM Specimen Preparation Team Leica Microsystems

EM Specimen Preparation Hernalser Hauptstr. 219 A 1170 Vienna Austria

List of abbreviations

2

List of abbreviations Fix.: Fixation

GAD: Glutaraldehyde

HPF: High Pressure Freezing

LTE: Low Temperature Embedding

ON: overnight

pFAD: para formaldehyde

PLT: Progressive Lowering of Temperature

Prog.: Program

RT: Room Temperature

Temp.: Temperature

Remarks and Disclaimer Please note: the protocols in this brochure are suggestions. Neither for the original protocols published by the original authors nor for the “Our proposal for the AFS” and “Input” protocols can we take any responsibility for the success of the users work. It was not our intention to give an overview to the field. This simply would have been impossible. When searching references for “freeze substitution” in a standard database one achieves more than 650 results! We neither wanted to give an introduction to the method – there are already very good books and reviews, e.g. Maunsbach & Afzelius (1999), Glauert & Lewis (1998), Parthasarathy (1995), Sitte et al. (1994), Steinbrecht & Müller (1987). Reviews of the method are given e.g. by Hermann & Müller (1997) for SEM and Howard & O´Donnell (1987), Hippe-Sanwald (1993), Nicolas & Bassot (1993) and Skepper (2000) for TEM. “State of the art” experimental surveys of different substitution media are given for TEM immunocytochemistry by Monaghan et al. (1998), for morphological TEM studies by Porta & Lopez-Iglesias (1998), for PLT by Robertson et al. (1992), for SEM (of Nematodes) by Wharton (1991) and for X-ray microanalysis by Palsgard et al. (1994). Even LM can be done after FS (e.g. He & Wetzstein 1997).

Rules of thumb

3

Rules of thumb • Around -80°C (depending on your specimen) recrystallisation will occur. Stay

≤ -85°C to be on the safe side. • Substitution time can be too short, but never too long. • Any slope between +4°C to +15°C for a FS will be sufficient for a good result –

try +5°C. • If you have no embedding / polymerisation protocol of your own for your resin

– keep to the resin manufacturers manual. If you are successful with your protocol – “Never change a winning team!”

• You can infiltrate with epoxy resin at room temperature (RT, i.e. +20°C) in the AFS, but we recommend to do this in a fume cupboard - your AFS is then free for the next FS run.

• You can heat polymerise epoxy resin in the AFS (e.g. at +60°C), but if you put it into an oven, your AFS is free for the next FS run.

• Monomers of acrylic resins are aggressive chemicals. If possible, keep the infiltration time as short as possible.

How to read the protocols:

• For convenience we use °C instead of K. • We assume room temperature (RT) of the original protocols to be +20°C in

“Our proposal for the AFS” and the “Input” protocols. • We assume overnight (ON) of the original protocols to be 12 hours in the “Our

proposal for the AFS” and the “Input” protocols. • FS media like “acetone” in the protocols are always “dry” media, e.g.

anhydrous acetone, dried with molecular sieve or CaCl2. • Empty lines in the column “Reagent” indicate NO CHANGE to the existing

condition, e.g.

1 Acetone containing 2% OsO4 72 -85 2 13 +5 3

indicates that steps 2 and 3 are also in acetone containing 2% OsO4.

• Empty fields in “Time”, “Temp.” or “Slope” in the original protocols indicate a lack of information in the original “Material & Methods” section. We filled these gaps with values that seemed reasonable to us in the “Our proposal for the AFS” and “Input” protocols.

• ! indicates that in this program polymerisation takes place under UV. We decided to put the polymerisation step into a program of its own (even if it would fit into the previous program). This gives the user the possibility to have a “polymerisation program” and modify polymerisation values without affecting the FS program steps.

• We assume that you do infiltration, embedding and heat polymerisation with epoxy resins in the AFS and therefore are giving time, slope and temperature for this whenever possible in the “Our proposal for the AFS” and “Input” protocols. However, you can do this in a fume cupboard and an oven respectively to have your AFS free for the next FS run.

• When the original authors gave no detailed information on the exact embedding schedule (e.g. just “embedding in Epon”) we substituted this by a schedule that seemed reasonable to us. For a few protocols this was not

How to read the protocols

4

possible. Please contact the corresponding resin manufacturer to receive more information.

• To some original protocols we have attached citations of methodical important hints and comments by the original authors.

The classic FS protocol by Van Harreveld & Crowell (1964) can be seen as “the mother of all routine freeze substitutions” for TEM. To illustrate our transcription from the original Material and Methods section to the “Input“protocol for the AFS we show it here: “Two freezers were used, a food freezer kept at -25°C and a “Revco” Ultra-low temperature freezer (SZC 859) rated to a minimum temperature of -95°C. Substitution fixation was carried out at -25°C, -50°C and -85°C in 2% osmium tetroxide in acetone, methyl- or ethyl-alcohol. The tissues were kept routinely in these solvents for three days. The temperature was then increased in steps (1 hour at -25°, 1 hour at 0°) to room temperature. The material tends to dissolve gas at low temperature which is released and can lead to bubble formation when the tissue is suddenly brought to room temperature. […] The material was embedded in the epoxy resin Maraglas (Freeman and Spurlock, `62),” Van Harreveld A. & Crowell, J. (1964) Kerckhoff Laboratories of the Biological Sciences California Institute of Technology, Pasadena, California, USA

Morphological study Specimen: Mouse (liver)

Fix.: Impact freezing

Step Reagent Time [h]

Temp. [°C]

Slope [°Ch-1]

1 Acetone* containing 2% OsO4 72 -85 2 1 -25 3 1 0 4 RT 5 Embedding in Maraglas

* alternatively: methanol or ethanol containing 2% OsO4. Van Harreveld & Crowell (1964): “[…] the best results were obtained by substitution fixation in 2% OsO4 in acetone at -85°C. When the substitution was carried out at -50°C reasonable preparations were still obtained. No passable preparations were obtained by substitution at -25°C. With methyl- and ethyl-alcohol as solvents, the results were poor when the fixation was carried out at -50°C; some passable blocks were obtained at -85°C.”

How to read the protocols

5

Our proposal for the AFS: Prog. Step Reagent Time

[h] Temp.

[°C] Slope [°Ch-1]

1 T1 Acetone* containing 2% OsO4 72 -85 S1 1 +60 T2 0.1 -25 S2 1 +25 T3 0.1 0

2 T1 Rinse in acetone 4 x 15 min 28 +20 Epon**:acetone 1:2 (3 h) Epon:acetone 2:1 (12h) pure Epon (12h) pure Epon + 1.5% BDMA S1 1 +40 T2 Polymerisation 48 +60

**instead of “Maraglas” we recommend to use Epon. Input:

Step Time [h]

Temp. [°C]

Slope [°Ch-1]

T1 72 -85 S1 1 +60 T2 0.1 -25 S2 1 +25

1

T3 0.1 0 2 T1 28 +20

S1 1 +40 T2 48 +60 S2 0 0 T3 0 +60

Various Tissues and Cells

6

Sample Protocols LTE for TEM Various Tissues and Cells Studer D., pers. com. Anatomisches Institut University of Bern Bühlstr. 26 CH-3000 Bern 9, Switzerland

Morphological study Specimen: Yeast, Rat (liver, kidney, brain, nerve), Ivy Hedera helix (leaf)

Fix.: HPF in Leica EM PACT

Step Reagent Time

[h] Temp.


1 Acetone containing 2% OsO4 26 -90 2 15 +2 3 8 -60 4 15 +2 5 8 -30 6 Acetone 100% 0.5 -30 7 Acetone 100% 0.5 -30 8 Epon:acetone 1:2 3 +4 9 Epon:acetone 2:1 ON RT

10 pure Epon ON +20 11 pure Epon + 1.5% BDMA 12 Polymerisation 72 +60

Our proposal for the AFS:

Prog. Step Reagent Time [h]

Temp. [°C]

Slope [°Ch-1]

1 T1 Acetone containing 2% OsO4 26 -90 S1 15 +2 T2 8 -60 S2 15 +2 T3 (8h) 9 -30 Acetone 100% (0.5h) Acetone 100% (0.5h)

2 T1 Epon:acetone 1:2 3 +4 S1 0.5 +32 T2 Epon:acetone 2:1 (12h) 24 +20 pure Epon (12h) pure Epon + 1.5% BDMA S2 1 +40 T3 Polymerisation 72 +60

Various Tissues and Cells

7

Input: Step Time

[h] Temp.


T1 26 -90 S1 15 +2 T2 8 -60 S2 15 +2

1

T3 9 -30 2 T1 3 +4

S1 0.5 +32 T2 24 +20 S2 1 +40 T3 72 +60

Plant Tissue Studer D. & Graber W., pers. com. Anatomisches Institut University of Bern Bühlstr. 26 CH-3000 Bern 9, Switzerland

Morphological study Specimen: Rice, Oryza sativa (anthers)

Fix.: HPF in Leica EM PACT


Temp. [°C]

Slope [°Ch-1]

1 Acetone containing 2% OsO4 26 -90 2 15 +2 3 8 -60 4 15 +2 5 5 -30 6 Acetone 100% 1 -30 7 Acetone 100% 1 -10 8 Epon:acetone 1:2 2 +4 9 Epon:acetone 2:1 2 +20

10 pure Epon + 1.5% BDMA ON +20 11 pure Epon + 1.5% BDMA 5 +20 12 pure Epon + 1.5% BDMA +20 13 Polymerisation 72 +60

Plant Tissue

8


[h] Temp.


1 T1 Acetone containing 2% OsO4 26 -90 S1 15 +2 T2 8 -60 S2 15 +2 T3 (5h) 6 -30 Acetone 100%(1h)

2 T1 Acetone 100% 1 -10 S1 0.5 +28 T2 Epon:acetone 1:2 2 +4 S2 0.5 +32 T3 Epon:acetone 2:1 (2h) 19 +20 pure Epon + 1.5% BDMA (12h) pure Epon + 1.5% BDMA (5h) pure Epon + 1.5% BDMA

3 T1 Polymerisation 72 +60 Input:

Step Time [h]

Temp. [°C]

Slope [°Ch-1]

T1 26 -90 S1 15 +2 T2 8 -60 S2 15 +2

1

T3 6 -30 2 T1 1 -10

S1 0.5 +28 T2 2 +4 S2 0.5 +32 T3 19 +20

3 T1 72 +60 S1 0 0 T2 0 +60 S2 0 0 T3 0 +60

Plant Tissue

9

Cole L. et al. (2000) School of Biological Sciences, University of New South Wales, Sydney, NSW, 2052 Australia

Morphological study Specimen: Fungus (Mycelium)

Fix.: Plunge freezing.


Temp. [°C]

Slope [°Ch-1]

1 Acetone containing 2% OsO4 72 -85 2 13 +5 3 2 -20 4 3.2 +5 5 2 -4 6 4.8 +5 7 2 RT 8 Rinse in acetone 4 x 15 min 9 Quetol 651* in acetone 5% 1 RT

10 “ 10% 1 RT 11 “ 20% 1 RT 12 “ 30% 1 RT 13 “ 40% 2 RT 14 “ 60% 2 RT 15 “ 60% ON RT 16 “ 80% 2 RT 17 Quetol 651 100% 1 RT 18 Quetol 651 100% RT 19 Polymerisation 14 +60

* according to Howard & O´Donnell (1987) Howard & O´Donnell (1987): “Specimens must be infiltrated very slowly, especially at the initial step […]. With walled cells, poor infiltration after freeze substitution is a common problem that can be avoided by using extra care […].”

Plant Tissue

10


[h] Temp.


T1 Acetone containing 2% OsO4 72 -85 S1 13 +5 T2 2 -20 S2 3.2 +5

1

T3 2 -4 T1 0 -4 S1 4.8 +5 T2 (2h) 27 +20

2

Rinse in acetone 4 x 15 min Quetol 651 in acetone 5% (1h) “ 10% (1h)

“ 20% (1h) “ 30% (1h) “ 40% (2h)

“ 60% (2h) “ 60% (12h) “ 80% (2h)

Quetol 651 pure (1h) Quetol 651 pure (1h)

S2 1 +40

T3 Polymerisation 14 +60

Input:

Prog. Step Time [h]

Temp. [°C]

Slope [°Ch-1]

T1 72 -85 S1 13 +5 T2 2 -20 S2 3.2 +5

1

T3 2 -4 T1 0 -4 S1 4.8 +5 T2 27 +20 S2 1 +40

2

T3 14 +60

Plant Tissue

11

Wang, Y. et al. (2000) Department of Biological Sciences Michigan Technological University Houghton Michigan 49931, USA

Morphological study Specimen: diatomes

Fix.: HPF

Step Reagent Time

[h] Temp.

[°C]

1 Acetone containing 1% OsO4 72 -80 2 8 -20 3 1 0 4 1 RT 5 Rinse repeatedly in acetone RT 6 Spurr:acetone 1:3 ON RT 7 Spurr:acetone 1:1 4 RT 8 Spurr:acetone 3:1 4 RT 9 Spurr 100% 24 RT

10 Spurr 100% 24 RT 11 Polymerisation 24 +60



Temp. [°C]

Slope [°Ch-1]

1 T1 Acetone containing 1% OsO4 72 -80 S1 12 +5 T2 8 -20 S2 4 +5 T3 1 0

2 T1 (1h) 69 +20 Rinse repeatedly in acetone Spurr:acetone 1:3 (12h) Spurr:acetone 1:1 (4h) Spurr:acetone 3:1(4h) Spurr 100% (24h) Spurr 100% (24h) S1 1 +40 T2 Polymerisation 24 +60

Plant Tissue

12

Input : Prog. Step Time

[h] Temp.


T1 72 -80 S1 12 +5 T2 8 -20 S2 4 +5

1

T3 1 0 T1 69 +20 S1 1 +40 T2 24 +60 S2 0 0

2

T3 0 +60 Bourett, T.M. et al. (1999) DuPont Agricultural Products P.O. Box 80402 Wilmington, DE 19880-0402 USA

Morphological study Specimen: Rice, Oryza sativa (leaves)

Fix.: HPF

Step Reagent Time

[h] Temp.


1 Acetone containing 2% OsO4 58 -90 2 6 +5 3 12 -60 4 6 +5 5 6 -30 6 0.5 +4 7 Embedding in Epon 812

according to Studer et al. 1995

Bourett, T.M. et al. (1999): “[…], cellular membranes were poorly stained, a problem often associated with freeze substitution (Hoch 1986).”

Plant Tissue

13



Temp. [°C]

Slope [°Ch-1]

T1 Acetone containing 2% OsO4 58 -90 S1 6 +5 T2 12 -60 S2 6 +5

1

T3 6 -30 T1 0.5 +4 2 S1 30% Epon in acetone 3.2 +5

T2 (5h) 85 +20 70% Epon in acetone (8h) +20 100% Epon (72h) 100% Epon with accelerator S2 2 +20 T3 Polymerisation 120 +60

Input:

Step Time [h]

Temp. [°C]

Slope [°Ch-1]

T1 58 -90 S1 6 +5 T2 12 -60 S2 6 +5

1

T3 6 -30 2 T1 0.5 +4

S1 3.2 +5 T2 85 +20 S2 2 +20 T3 120 +60

Plant Tissue

14

Derksen, J. et al. (1999) Department of Experimental Botany Catholic University of Nijmegen Toernooiveld 1 6525 ED Nijmegen, The Netherlands

Morphological study Specimen: Pinus (pollen)

Fix.: Plunge freezing

Step

Reagent Time

[h] Temp.


1 Acetone containing 2% OsO4 ,

0.1% uranyl acetate 36 -90

2 27 +4 3 Rinse in acetone RT 4 Embedding in Spurr


Step

Reagent Time [h]

Temp. [°C]

Slope [°Ch-1]

T1 Acetone containing 2% OsO4 ,

0.1% uranyl acetate 36 -90

S1 27 +4 T2 Rinse in acetone 68 +20

1

Spurr:acetone 1:3 (12h) Spurr:acetone 1:1 (4h) Spurr:acetone 3:1(4h) Spurr 100% (24h) Spurr 100% (24h) S2 1 +40 T3 Polymerisation 24 +60

Input:

Step

Time [h]

Temp. [°C]

Slope [°Ch-1]

T1 36 -90 S1 27 +4 T2 68 +20 S2 1 +40

1

T3 24 +60

Plant Tissue

15

Harding et al. (1999) Department of Plant Biology Arizona State University P.O. Box 871601 Tempe AZ 85287-1601, USA

Morphological study Specimen: Phaseolus(leaf),

Uromyces (fungus) Fix.: HPF

Step

Reagent Time

[h] Temp.


1 Acetone containing 2% OsO4

and 0.05% uranyl acetate 72 -85

2 12 +8.75 3 Wash in acetone 3 x 15 min RT 4 Embedding in Epon-Araldit 5 Polymerisation 48 +70


Step

Reagent Time [h]

Temp. [°C]

Slope [°Ch-1]

T1 Acetone containing 2% OsO4


S1 12 +8.75 T2 Wash in acetone 3 x 15 min 9.75 +20

1

Epon-Araldite in acetone 30% (3h) Epon-Araldite in acetone 70% (3h) Epon-Araldite in acetone 100% (3h) S2 1 +50 T3 Polymerisation 48 +70

Input:

Step

Time [h]

Temp. [°C]

Slope [°Ch-1]

T1 72 -85 S1 12 +8.75 T2 9.75 +20 S2 1 +50

1

T3 48 +70

Plant Tissue

16

Lonsdale, J. E. et al. (1999) Department of Plant and Microbial Biology and Electron Microscope Laboratory University of California, Berkeley CA 94720 3102, USA

Morphological study, Immunocytochemistry

Specimen: Barley, Hordeum vulgare (protoplasts)

Fix.: HPF

Alternative 1: Morphological study


Temp. [°C]

Slope [°Ch-1]


0.2% uranyl acetate 96 -78.5

2 10 +5.85 3 12 -20 4 4 +10 5 Wash 3 x in acetone (10 min) RT 6 Epon*:acetone 1:2 4 RT 7 Epon*:acetone 1:1 1 RT 8 Epon*:acetone 2:1 1 RT 9 Epon* 100% 1 RT

10 Epon* containing accelerator ON RT 11 Polymerisation 48 +60

*special non-compressible mixture Alternative 2: Immunocytochemistry


Temp. [°C]

Slope [°Ch-1]

1 Acetone containing 0.2% GAD,


2 10 +5.85 3 12 -20 4 4 +10 5 Wash 3 x in acetone (10 min) RT 6 LR White:acetone 1:2 for 15 min +45 7 LR White:acetone 1:1 for 15 min +45 8 LR White:acetone 2:1 for 15 min +45 9 LR White 100% for 15 min +45

10 LR White 100% for 15 min +45 11 Polymerisation** for 45 min +80

** embedding in microwave oven according to Giberson et al. 1997

Plant Tissue

17

Our proposal for the AFS : Alternative 1: Morphological study


Temp. [°C]

Slope [°Ch-1]

T1 Acetone containing 0.2% GAD,


S1 10 +5.85 T2 12 -20 S2 4 +10

1

T3 Wash in acetone (3 x 10 min) 20.5 +20 Epon:acetone 1:2 (4h) Epon:acetone 1:1 (1h)

Epon:acetone 2:1 (1h) Epon:acetone 2:1 (1h) Epon 100% (1h)

Epon containing accelerator (12h) T1 Polymerisation 48 +60

Input :

Step Time [h]

Temp. [°C]

Slope [°Ch-1]

T1 96 -78.5 S1 10 +5.85 T2 12 -20 S2 4 +10

1

T3 20.5 +20 2 T1 48 +60

S1 0 0 T2 0 +60 S2 0 0 T3 0 +60

Plant Tissue

18

Alternative 2: Immunocytochemistry


Temp. [°C]

Slope [°Ch-1]



S1 10 +5.85 T2 12 -20 S2 4 +10

1

T3 Wash in acetone (3 x 10 min) 0.5 +20 T1 LR White:acetone 1:2 for 15 min 1.2 +45

LR White:acetone 1:1 for 15 min

LR White:acetone 2:1 for 15 min LR White 100% for 15 min LR White 100% for 15 min

Polymerisation** for 45 min Input:

Step Time [h]

Temp. [°C]

Slope [°Ch-1]

T1 96 -78.5 S1 10 +5.85 T2 12 -20 S2 4 +10

1

T3 0.5 +20 2 T1 1.2 +45

S1 0 0 T2 0 +45 S2 0 0 T3 0 +45

Staff, I. A. et al. (1999) School of Botany, The University of Melbourne Parkville, Victoria, Australia

Immunocytochemistry Specimen: Ryegrass, Lolium perenne

(pollen) Fix.: Plunge freezing

Step Reagent Time

[h] Temp.

[°C]

1 2,2 dimethoxypropane containing 0.5% GAD,

0.5% pFAD, 10% ethanol, 10% methanol 168 -80

2 RT 3 Rinse 4 Incubate in 10% dimethylsulfoxide 1 5 Embed in LR Gold + 1% benzil under a lamp 24 -25

Staff, I. A. et al. (1999): “Incubation in dimethylsulfoxide prior to embedding in LR Gold resin provided good infiltration with resin embedded material.”

Plant Tissue

19



Temp. [°C]

Slope [°Ch-1]

T1 2,2 dimethoxypropane containing 0.5% GAD,

0.5% pFAD, 10% ethanol, 10% methanol 168 -80

S1 20 +5 T2 Rinse 1 +20

1

Incubate in 10% dimethylsulfoxide (1h) T1 Embed in LR Gold + 1% benzil under a lamp 24 -25 !2

According to the manufacturers manual

Input:

Step Time [h]

Temp. [°C]

Slope [°Ch-1]

T1 168 -80 S1 20 +5 T2 1 +20 S2 0 0

1

T3 0 +20 !2 T1 24 -25

S1 0 0 T2 0 -25 S2 0 0 T3 0 -25

Porta, D. & Lopez-Iglesias, C. (1998) Laboratori de Botanica Facultat de Farmacia Universitat de Barcelona Joan XXIII s/n, 08028 Barcelona, Spain

Morphological study Specimen: green alga, Jaagiella

Fix.: Plunge freezing, Impact freezing

Alternative 1: Acetone substitution


Temp. [°C]

Slope [°Ch-1]

1 Acetone 72 -90 2 Acetone* -90 3 14 +5 4 -20 5 +4 6 Rinse in acetone 3 - 4 RT 7 Embedding in Spurr RT 8 Polymerisation 48 +60

* alternatively: acetone containing 3% GAD; acetone containing 1 – 3% OsO4; acetone containing 0.5% uranyl acetat; acetone containing 0.1% uranyl acetate and 2% OsO4

Plant Tissue

20

Alternative 2: Acetone with 3% GAD followed by 1% OsO4 at 4°C


Temp. [°C]

Slope [°Ch-1]

1 Acetone 72 -90 2 Acetone containing 3% GAD -90 3 14 +5 4 -20 5 Acetone containing 2% OsO4 1 +4 6 Rinse in acetone 3 - 4 RT 7 Embedding in Spurr RT 8 Polymerisation 48 +60

Alternative 3: Methanol substitution


Temp. [°C]

Slope [°Ch-1]

1 Methanol 72 -90 2 Methanol* -90 3 14 +5 4 -20 5 +4 6 Rinse in methanol 3 - 4 RT 7 Embedding in Spurr RT 8 Polymerisation 48 +60

* alternatively: methanol containing 3% GAD; methanol containing 1 – 3% OsO4 Porta, D. & Lopez-Iglesias, C. (1998): “Acetone gave better results than methanol as a substitution solvent when high temperature embedding was performed. The best cryosubstitution for ultrastructural studies was that in which osmium tetroxide or a mixture of osmium tetroxide and uranyl acetate was used.” Our proposal for the AFS: Alternative 1: Acetone substitution


Temp. [°C]

Slope [°Ch-1]

T1 Acetone (72h) 84 -90

Acetone* (12h) S1 14 +5

1

T2 12 -20 S2 4.8 +5 T3 12 +4

T1 Rinse in acetone (4h) 72 +20 2 Spurr:acetone 1:3 (12h)

Spurr:acetone 1:1 (4h) Spurr:acetone 3:1(4h) Spurr 100% (24h) Spurr 100% (24h) S1 1 +40 T2 Polymerisation 24 +60

Plant Tissue

21

Input: Step Time

[h] Temp.


T1 84 -90 S1 14 +5 T2 12 -20 S2 4.8 +5

1

T3 12 +4 2 T1 72 +20

S1 1 +40 T2 24 +60 S2 0 0 T3 0 +60

Alternative 2: Acetone with 3% GAD followed by 1% OsO4 at 4°C


Temp. [°C]

Slope [°Ch-1]

1 T1 Acetone (72h) 84 -90 Acetone containing 3% GAD (12h) S1 14 +5 T2 12 -20 S2 4.8 +5 T3 Acetone containing 2% OsO4 1 +4 2 T1 Rinse in acetone (4h) 72 +20 Spurr:acetone 1:3 (12h) Spurr:acetone 1:1 (4h) Spurr:acetone 3:1(4h) Spurr 100% (24h) Spurr 100% (24h) S1 1 +40 T2 Polymerisation 24 +60

Input:

Step Time [h]

Temp. [°C]

Slope [°Ch-1]

T1 84 -90 S1 14 +5 T2 12 -20 S2 4.8 +5

1

T3 1 +4 2 T1 72 +20

S1 1 +40 T2 24 +60 S2 0 0 T3 0 +60

Plant Tissue

22

Alternative 3: Methanol substitution Prog. Step Reagent Time

[h] Temp.


1 T1 Methanol (72h) 84 -90 Methanol* (12h) S1 14 +5 T2 12 -20 S2 4.8 +5 T3 12 +4

2 T1 Rinse in methanol (4h) 72 +20 Spurr: methanol 1:3 (12h) Spurr: methanol 1:1 (4h) Spurr: methanol 3:1(4h) Spurr 100% (24h) Spurr 100% (24h) S1 1 +40 T2 Polymerisation 24 +60

* alternatively: methanol containing 3% GAD; methanol containing 1 – 3% OsO4 Input:

Step Time [h]

Temp. [°C]

Slope [°Ch-1]

T1 84 -90 S1 14 +5 T2 12 -20 S2 4.8 +5

1

T3 12 +4 2 T1 72 +20

S1 1 +40 T2 24 +60 S2 0 0 T3 0 +60

Plant Tissue

23

Tiedemann, J. et al. (1998) Botanisches Institut Lehrstuhl II Christian Albrechts Universität Olshausenstr. 40 D-24098 Kiel, Germany

Morphological study Specimen: Potato, Solanum tuberosum

(protoplasts) Solanum nigrum Fix.: HPF

Step Reagent

Time [h]

Temp. [°C]

Slope [°Ch-1]

1 Acetone containing 0.5% uranyl acetate 14 -90 2 Wash in acetone 3 Acetone 1 +30 4 Acetone containing 1% OsO4 8 -60 5 8 -30 6 Wash 3 x in acetone 8 -30 7 Spurr without accelerator 2 +25 8 60 RT 9 Addition of accelerator RT

Tiedemann, J. et al. (1998): “For the very slow resin infiltration of HPF samples Spurr´s resin without any accelerator was used according to Parthasarathy (1995). […] Using the complete resin mixture from the first infiltration step prevented adequate resin infiltration.” Our proposal for the AFS:


Temp. [°C]

Slope [°Ch-1]

T1 Acetone containing 0.5% uranyl acetate 14.5 -90

Wash in acetone (3 x 10 min) S1 Acetone 1 +30

1

T2 Acetone containing 1% OsO4 8 -60 S2 1 +30 T3 8 -30

2 T1 Wash 3 x in acetone 8 -30 S1 Spurr without accelerator 2 +25 T2 60 +20 Addition of accelerator +20 Polymerisation according to the manu-

facturers manual

Plant Tissue

24

Input: Step Time

[h] Temp.


T1 14.5 -90 S1 1 +30 T2 8 -60 S2 1 +30

1

T3 8 -30 2 T1 8 -30

S1 2 +25 T2 60 +20 S2 0 0 T3 0 +20

Thijssen M.T. et al. (1998) Laboratory of Plant Cytology and Morpholgy Graduate School of Experimental Plant sciences Wageningen Agricultural University Arboretumlaan 4 NL-6703 BD Wageningen, The Netherlands

Morphological study Specimen: Petunia (ovules)

Fix.: HPF

Step Reagent Time

[h] Temp.

[°C]


And 0.1% uranyl acetate 8 -90

2 8 -60 3 8 -30 4 Rinse in acetone RT 5 Embedding in Spurr

Thijssen M.T. et al. (1998): “Because of its insolubility in acetone at -90°C, the 1 – hexadecene has to be removed from the sample surface before freeze-substitution, in order to have sufficient exchange between ice and the substitution medium (Hohenberg et al 1994).”

Plant Tissue

25



Temp. [°C]

Slope [°Ch-1]



S1 6 +5 T2 8 -60 S2 6 +5

1

T3 8 -30 2 T1 Rinse in acetone (1h) 69 +20

Spurr:acetone 1:3 (12h) Spurr:acetone 1:1 (4h) Spurr:acetone 3:1 (4h) Spurr 100% (24h) Spurr 100% (24) S1 1 +40 T2 Polymerisation 24 +60

Input:

Step Time [h]

Temp. [°C]

Slope [°Ch-1]

T1 8 -90 S1 6 +5 T2 8 -60 S2 6 +5

1

T3 8 -30 2 T1 69 +20

S1 1 +40 T2 24 +60 S2 0 0 T3 0 +60

Plant Tissue

26

Takahashi, H. (1997) Department of Biological Sciences Graduate School of Science University of Tokyo, Hongo Tokyo 113, Japan

Immunocytochemistry Specimen: Red alga (Cyanidium

caldarium) Fix.: Plunge freezing

Alternative 1:


Temp. [°C]

1 Acetone 48 -80 2 2 -20 3 2 0 4 2 +4 5 2 RT 6 Wash 2 x in acetone 7 Embedding in LR White,

Alternatively: Spurr +70 °C 48 +50

Alternative 2: Step 1 Acetone containing 1% GAD, following steps as above Alternative 3: Step 1 Acetone containing 1% OsO4, following steps as above Takahashi, H. (1997): “[…] results indicate that the rapid freeze substitution in absolute acetone and embedding in LR White resin is the optimal approach for immunogold labelling.” Our proposal for the AFS:


Temp. [°C]

Slope [°Ch-1]

T1 Acetone 48 -80 S1 12 +5 T2 2 -20 S2 4 +5

1

T3 2 0 T1 2 +4 2 S1 3.2 +5

T2 (2h) 24.5 +20 Wash in acetone (2 x 15 min) Spurr:acetone 1:3 (8h) Spurr:acetone 1:1 (4h) Spurr:acetone 3:1(4h) Spurr 100% (4h) Spurr 100% (4h) S2 1 +40 T3 Polymerisation 24 +70

Plant Tissue

27

Input: Step Time

[h] Temp.


T1 48 -80 S1 12 +5 T2 2 -20 S2 4 +5

1

T3 2 0 2 T1 2 +4

S1 3.2 +5 T2 24.5 +20 S2 1 +40 T3 24 +70

For the embedding in LR White please read the resin manufacturers manual. Czymmek, K.J. et al. (1996) DuPont Company Science and Engeneering Laboratories Wilmington, DE, USA

Immunocytochemistry Specimen: Fungus (hyphae)


Step Reagent Time

[h] Temp.

[°C]

1 Ethanol containing 2 % pFAD 72 -80 2 2 RT 3 Rinse 3 x in ethanol RT 4 Methacrylate:ethanol1:2 RT 5 Methacrylate:ethanol1:1 RT 6 Methacrylate:ethanol2:1 RT 7 Methacrylate 100 % RT 8 Methacrylate 100 % Overn. +4 9 Polymerisation under UV Overn.

Plant Tissue

28


[h] Temp.


T1 Ethanol containing 2 % pFAD 72 -80 S1 20 +5 T2 Ethanol containing 2 % pFAD (2h) 18 +20

Methacrylate:ethanol1:2 (4h)

1

Methacrylate:ethanol1:1 (4h) Methacrylate:ethanol2:1 (4h) Methacrylate 100 % (4h) S2 4 -4 T3 Methacrylate 100 % 12 +4

!2 T1 Polymerisation under UV 12 +4 Input:

Step Time [h]

Temp. [°C]

Slope [°Ch-1]

T1 72 -80 S1 20 +5 T2 18 +20 S2 4 -4

1

T3 12 +4 !2 T1 12 +4

S1 0 0 T2 0 +4 S2 0 0 T3 0 +4

Plant Tissue

29

Ristic Z. & Ashworth E.N. (1993) Centre for Plant Environmental Stress Physiology Department of Horticulture Purdue University West Lafayette, IN 47907 USA

Morphological study Specimen: Cornus spp. (wood)


Step Reagent Time

[h] Temp.

[°C]

1 Methanol containing 2 % OsO4 112 -80 2 Methanol containing 2 % OsO4 112 -80 3 Methanol containing 2 % OsO4 112 -80 4 24 -15 5 24 +4 6 3 RT 7 Methanol 100 % 0.5 RT 8 Methanol 80 % 0.5 RT 9 Methanol 60 % 0.5 RT

10 Methanol 40 % 0.5 RT 11 Methanol 20 % 0.5 RT 12 Distilled water 0.5 RT 13 25 mM sodium phosphate buffer pH 6.9 48 RT 14 Acetone 30 % 2 RT 15 Acetone 50 % 2 RT 16 Acetone 70 % 2 RT 17 Acetone 80 % 2 RT 18 Acetone 90 % 2 RT 19 Acetone 100 % 2 RT 20 Rinse 2 x in acetone RT 21 Add acidified 2,2-dimethoxypropane (DMP) RT 22 20 % Spurr, 77 % acetone, 3 % DMP 8 RT 23 40 % Spurr, 57 % acetone, 3 % DMP 10 RT 24 60 % Spurr, 37 % acetone, 3 % DMP 11 RT 25 80 % Spurr, 17 % acetone, 3 % DMP 13 RT 26 100 % Spurr 12 RT 27 100 % Spurr 24 RT 28 Polymerisation 24 60

Plant Tissue

30


[h] Temp.


T1 Methanol containing 2 % OsO4

(3 x 112 h) 336 -80

S1 13 +5 T2 24 -15 S2 3.8 +5

1

T3 24 +4 2 T1 (3h) 144 +20 Methanol 100% (0.5h) Methanol 80 % (0.5h) Methanol 60 % (0.5h) Methanol 40 % (0.5h) Methanol 20 % (0.5h) Distilled water (0.5h) 25 mM sodium phosphate buffer pH 6.9 (48h) Acetone 30 % (2h) Acetone 50 % (2h) Acetone 70 % (2h) Acetone 80 % (2h) Acetone 90 % (2h) Acetone 100 % (2h) Rinse 2 x in acetone Add acidified 2,2-dimethoxypropane (DMP) 20 % Spurr, 77 % acetone, 3 % DMP (8h) 40 % Spurr, 57 % acetone, 3 % DMP (10h) 60 % Spurr, 37 % acetone, 3 % DMP (11h) 80 % Spurr, 17 % acetone, 3 % DMP (13h) 100 % Spurr (12h) 100 % Spurr (24h) S1 1 +40 T2 Polymerisation 24 +60

Input:

Step Time [h]

Temp. [°C]

Slope [°Ch-1]

T1 336 -80 S1 13 +5 T2 24 -15 S2 3.8 +5

1

T3 24 +4 2 T1 144 +20

S1 1 +40 T2 24 +60 S2 0 0 T3 0 +60

Animal/Human Tissue

31

Animal/Human Tissue Ap Gwynn, I. et al. (2000) Institute of Biological Sciences The University of Wales Aberystwyth, Wales SY23 3DA United Kingdom

Morphological study Specimen: Rabbit (cartilage)


Step Reagent Time

[h] Temp.


1 Acetone containing 2% acrolein 192 -80 2 20 +5 3 Acetone containing 1% OsO4 -30 4 10 +5 5 Rinse in acetone +20 6 Embedding in K11M, fresh resin daily 168 -30 7 Polymerisation under UV -30



Temp. [°C]

Slope [°Ch-1]

T1 Acetone containing 2% acrolein 192 -80 S1 20 +5 T2 Acetone containing 1% OsO4 12 -30 S2 10 +5

1

T3 Rinse in acetone 0.5 +20 T1 Embedding in K11M, fresh resin (7 x 24 h) 168 -30 2

Polymerisation under UV according to the manufacturers manual

-30

Input:

Step Time [h]

Temp. [°C]

Slope [°Ch-1]

T1 192 -80 S1 20 +5 T2 12 -30 S2 10 +5

1

T3 0.5 +20 2 T1 168 -30

S1 0 0 T2 0 -30 S2 0 0 T3 0 -30

Animal/Human Tissue

32

Young, R.D. et al. (2000) Connective Tissue Biology Laboratories School of Biosciences Cardiff, UK CF1 3US, UK

Immunocytochemistry Specimen: human (femoral articular

cartilage) Fix.: HPF



Temp. [°C]

Slope [°Ch-1]

1 Acetone containing 2% OsO4 48 -90 2 11 +10 3 Araldite:acetone 1:3 +20 4 Araldite:acetone 1:1 +20 5 Araldite:acetone 3:1 +20 6 Araldite pure, several changes +20 7 Polymerisation in fresh Araldite +60

Alternative 2: Methanol substitution


Temp. [°C]

Slope [°Ch-1]

1 Methanol 24 -90 2 4 +10 3 HM20:methanol 1:3 1 -50 4 HM20:methanol 1:1 1 -50 5 HM20:methanol 3:1 1 -50 6 HM20 pure, several changes -50 7 Polymerisation under UV 48 -50

Our proposal for the AFS: Alternative 1: Acetone substitution


Temp. [°C]

Slope [°Ch-1]

T1 Acetone containing 2% OsO4 48 -90 S1 11 +10

1

T2 Araldite:acetone 1:3 (4h) 24 +20 Araldite:acetone 1:1 (4h) Araldite:acetone 3:1 (4h)

several changes Araldite pure (3x4h) S2 1 +40 T3 Polymerisation in fresh Araldite 48 +60

Animal/Human Tissue

33

Input: Step Time

[h] Temp.


T1 48 -90 S1 11 +10 T2 24 +20 S2 1 +40

1

T3 48 +60 Alternative 2: Methanol substitution


Temp. [°C]

Slope [°Ch-1]

T1 Methanol 24 -90 S1 4 +10 T2 HM20:methanol 1:3 (1h) 6 -50

1

HM20:methanol 1:1 (1h) HM20:methanol 3:1 (1h) HM20 pure, several changes (3x1h)

!2 T1 Polymerisation under UV 48 -50 Input:

Step Time [h]

Temp. [°C]

Slope [°Ch1]

T1 24 -90 S1 4 +10 T2 6 -50 S2 0 0

1

T3 0 -50 !2 T1 48 -50

S1 0 0 T2 0 -50 S2 0 0 T3 0 -50

Animal/Human Tissue

34

Ameye, L. et al. (1999) Laboratory of Marine Biology CP 160/15 Free University of Brussels 50 Ave F.D. Roosevelt 1050 Bruxelles, Belgium

Immunocytochemistry Specimen: Sea urchin, Paracentrotus

lividus (skeletal tissue) Fix.: HPF



Temp. [°C]

1 Acetone containing 0.2% gallic

acid, 6% GAD 48 -90

2 12 -60 3 Wash 3 x in acetone -60 4 Acetone containing 2% OsO4 30 -60 5 8 -30 6 1 0 7 Wash 3 x in acetone 8 Epon/Araldite in acetone 30% 3 9 “ / “ in “ 70% 3

10 “ / “ in “ 100% 3 11 Polymerisation +60

Alternative 2: Ethanol substitution


Temp. [°C]

1 Ethanol containing 0.5% uranyl acetate 8 -90 2 8 -60 3 8 -30 4 1 0 5 Wash 3 x in acetone 6 LR Gold in acetone 33% 3 7 “ in “ 66% 12 8 LR Gold 100% 2 9 Polymerisation -20

Animal/Human Tissue

35

Our proposal for the AFS: Alternative 1: Acetone substitution


Temp. [°C]

Slope [°Ch-1]

T1 Acetone containing 0.2% gallic

acid, 6% GAD 48 -90

S1 6 +5 T2 (12h) 42.5 -60

1

Wash in acetone (3 x 10 min) Acetone containing 2% OsO4 (30h)

S2 6 +5 T3 8 -30 T1 0 -30 S1 6 +5

2

T2 (1h) 1.5 0 Wash in acetone (3 x 10 min)

S2 4 +5 T3 Epon/Araldite in acetone 30% (3h) 9 +20

“ 70% (3h) “ 100% (3h)


Step Time [h]

Temp. [°C]

Slope [°Ch-1]

T1 48 -90 S1 6 +5 T2 42.5 -60 S2 6 +5

1

T3 8 -30 2 T1 0 -30

S1 6 +5 T2 1.5 0 S2 4 +5 T3 9 +20

3 T1 48 +60 S1 0 0 T2 0 +60 S2 0 0 T3 0 +60

Animal/Human Tissue

36

Alternative 2: Ethanol substitution Prog. Step Reagent Time

[h] Temp. [°C]

Slope [°Ch-1]

T1 Ethanol containing 0.5% uranyl acetate 8 -90 S1 6 +5 T2 8 -60 S2 6 +5

1

T3 8 -30 T1 (1h) 18.5 0

Wash in acetone (3 x 10 min) 2

LR Gold in acetone 33% (3h) LR Gold in acetone 66% (12h) LR Gold 100% (2h) S1 4 -5

!3 T1 Polymerisation 48 -20 Input:

Step Time [h]

Temp. [°C]

Slope [°Ch-1]

T1 8 -90 S1 6 +5 T2 8 -60 S2 6 +5

1

T3 8 -30 2 T1 18.5 0

S1 4 -5 T2 48 -20 S2 0 0 T3 0 -20

!3 T1 48 -20 S1 0 0 T2 0 -20 S2 0 0 T3 0 -20

Animal/Human Tissue

37

Sawaguchi, A. et al. (1999) Department of Anatomy Miyazaki Medical College Miyazaki, Japan

Immunocytochemistry Specimen: Rat, stomach

Fix.: HPF

Step Reagent Time

[h] Temp.

[°C]

1 Acetone containing 0.5 % GAD 16 -80 2 Rinse in ethanol 3 x 10 min 0.5 -35 3 2 +18 4 Ethanol:K4M 1:1 1 -35 5 Ethanol:K4M 1:2 1 -35 6 K4M pure ON -35 7 Polymerisation under UV 24 -35 8 Polymerisation under UV 24 +18



Temp. [°C]

Slope [°Ch-1]

T1 Acetone containing 0.5 % GAD 16 -80 S1 9 +5 T2 Rinse in ethanol 3 x 10 min 0.5 -35 S2 10.6 +5

1

T3 2 +18 T1 Ethanol:K4M 1:1 (1h) 10 -35

Ethanol:K4M 1:2 (1h) 2

K4M pure (8h) T1 Polymerisation under UV 24 -35 S1 10.6 +5

!3

T2 Polymerisation under UV 24 +18

Animal/Human Tissue

38

Input:

Step Time [h]

Temp. [°C]

Slope [°Ch-1]

T1 16 -80 S1 9 +5 T2 0.5 -35 S2 10.6 +5

1

T3 2 +18 2 T1 10 -35

S1 0 0 T2 0 0 S2 0 -35 T3 0 0

!3 T1 24 -35 S1 10.6 +5 T2 24 +18 S2 0 0 T3 0 +18

Monaghan P. et al. (1998) Institute of Cancer Research 15, Cotswold Road, Sutton, Surrey SM2 5NG United Kingdom


Specimen: human (Lymphoma, breast cancer)

Mouse (heart, kidney, skeletal muscle, mammary gland)

Fix.: HPF

Alternative 1: Morphological study Step Reagent Time

[h] Temp.


1 Specimen* -160 2 14 +5 3 Acetone containing 2% OsO4 8 -90 4 8 -60 5 8 -30 6 Acetone RT

Embedding in Epon according to Monaghan et al. 1985

* alternatively directly to -90 °C. Monaghan P. et al. (1998): “Freeze-substitution in acetone containing 2% OsO4 followed by epoxy-resin embedding at room temperature gave optimum morphology.”

Animal/Human Tissue

39



Temp. [°C]

Slope [°Ch-1]

1 Methanol 36** -90 2 4 +10 3 -50 4 Ethanol 1 -50 5 Ethanol:HM20 3:1 1 -50 6 Ethanol:HM20 1:1 1 -50 7 Ethanol:HM20 1:3 1 -50 8 HM20 100% 1 -50 9 HM20 100% 1 -50

10 HM20 100% 18 -50 11 HM20 100% 1 x change -50 12 Polymerisation under UV 48 -50 13 7 +10 14 Polymerisation under UV 48 RT

** alternatively 24, 18, 8 hours. Alternative 3: Immunocytochemistry according to Palsgard et al. (1994)


Temp. [°C]

Slope [°Ch-1]

1 Tetrahydrofuran 48 -80 2 Ethanol 1 -50 3 Ethanol:HM20 3:1 1 -50 4 Ethanol:HM20 1:1 1 -50 5 Ethanol:HM20 1:3 1 -50 6 HM20 100% 1 -50 7 HM20 100% 1 -50 8 HM20 100% 18 -50 9 HM20 100% 1 x change -50

10 Polymerisation under UV 48 -50 11 7 +10 12 Polymerisation under UV 48 RT

Animal/Human Tissue

40

Alternative 4: Immunocytochemistry according to Edelmann (1991)


Temp. [°C]

Slope [°Ch-1]

1 Acetone 144 -90 2 4 +10 3 Ethanol 1 -50 4 Ethanol:HM20 3:1 1 -50 5 Ethanol:HM20 1:1 1 -50 6 Ethanol:HM20 1:3 1 -50 7 HM20 100% 1 -50 8 HM20 100% 1 -50 9 HM20 100% 18 -50

10 HM20 100% 1 x change -50 11 Polymerisation under UV 48 -50 12 7 +10 13 Polymerisation under UV 48 RT

Alternative 5 : Immunocytochemistry according to Kaneko & Walter (1995)


Temp. [°C]

Slope [°Ch-1]

1 Acetone 24 -90 2 2.5 +4 3 120 -80 4 5 +4 5 24 -60 6 Ethanol 1 -50 7 Ethanol:HM20 3:1 1 -50 8 Ethanol:HM20 1:1 1 -50 9 Ethanol:HM20 1:3 1 -50

10 HM20 100% 1 -50 11 HM20 100% 1 -50 12 HM20 100% 18 -50 13 HM20 100% 1 x change -50 14 Polymerisation under UV 48 -50 15 7 +10 16 Polymerisation under UV 48 RT

Monaghan P. et al. (1998): ”Following acetone substitution, samples were either infiltrated directly into HM20, which resulted in variable embedding quality, or transferred to ethanol at –50°C for 60 minutes prior to infiltration. This latter step was introduced as ethanol appears to be more compatible with Lowicryl HM20 when compared with acetone.”

Animal/Human Tissue

41

Our proposal for the AFS: Alternative 1: Morphological study


Temp. [°C]

Slope [°Ch-1]

1 T1 Specimen -160 S1 14 +5 T2 Acetone containing 2% OsO4 8 -90

S2 6 +5 T3 8 -60

2 T1 8 -30 S1 10 +5 T2 Acetone 8 +20 Embedding in Epon according to

Monaghan et al. 1985

Input:

Step Time [h]

Temp. [°C]

Slope [°Ch-1]

T1 -160 S1 14 +5 T2 8 -90 S2 6 +5

1

T3 8 -60 2 T1 8 -30

S1 10 +5 T2 8 +20 S2 0 0 T3 0 +20



Temp. [°C]

Slope [°Ch-1]

T1 Methanol 36 -90 S1 4 +10

1

T2 Ethanol (1h) 23 -50 Ethanol:HM20 1:1 (1h) Ethanol:HM20 1:3 (1h)

HM20 100% (1h) HM20 100% (1h) HM20 100% (18h)

HM20 100% 1 x change T1 Polymerisation under UV 48 -50 S1 7 +10

!2

T2 Polymerisation under UV 48 +20

Animal/Human Tissue

42

Input: Step Time

[h] Temp.


T1 36 -90 S1 4 +10 T2 23.1 -50 S2 0 0

1

T3 0 -50 !2 T1 48 -50

S1 7 +10 T2 48 +20 S2 0 0 T3 0 +20

Alternative 3: Immunocytochemistry according to Palsgard et al. (1994)


Temp. [°C]

Slope [°Ch-1]

T1 Tetrahydrofuran 48 -80 S1 6 +5

1

T2 Ethanol (1h) 23 -50 Ethanol:HM20 1:1 (1h) Ethanol:HM20 1:3 (1h)

HM20 100% (1h) HM20 100% (1h) HM20 100% (18h)


!2

T2 Polymerisation under UV 48 +20 Input:

Step Time [h]

Temp. [°C]

Slope [°Ch-1]

T1 48 -80 S1 6 +5 T2 23 -50 S2 0 0

1

T3 0 -50 !2 T1 48 -50

S1 7 +10 T2 48 +20 S2 0 0 T3 0 +20

Animal/Human Tissue

43

Alternative 4: Immunocytochemistry according to Edelmann (1991) Prog. Step Reagent Time

[h] Temp.


T1 Acetone 144 -90 S1 4 +10 T2 Ethanol (1h) 24 -50

1

Ethanol:HM20 3:1 (1h) Ethanol:HM20 1:1 (1h) Ethanol:HM20 1:3 (1h)

HM20 100% (1h) HM20 100% (1h) HM20 100% (18h)


!2

T2 Polymerisation under UV 48 +20 Input:

Step Time [h]

Temp. [°C]

Slope [°Ch-1]

T1 144 -90 S1 4 +10 T2 24 -50 S2 0 0

1

T3 0 -50 2 T1 48 -50

S1 7 +10 T2 48 +20 S2 0 0 T3 0 +20

Animal/Human Tissue

44

Alternative 5: Immunocytochemistry according to Kaneko & Walter (1995) Prog. Step Reagent Time

[h] Temp. [°C]

Slope [°Ch-1]

T1 Acetone 24 -90 S1 2.5 +4 T2 120 -80 S2 5 +4

1

T3 24 -60 2

T1 Ethanol (1h) 24 -50

Ethanol:HM20 3:1 (1h) Ethanol:HM20 1:1 (1h) Ethanol:HM20 1:3 (1h) HM20 100% (1h) HM20 100% (1h) HM20 100% (18h) HM20 100% 1 x change

!3 T1 Polymerisation under UV 48 -50 S1 7 +10 T2 Polymerisation under UV 48 +20

Input:

Step Time [h]

Temp. [°C]

Slope [°Ch-1]

T1 24 -90 S1 2.5 +4 T2 120 -80 S2 5 +4

1

T3 24 -60 2 T1 24 -50

S1 0 0 T2 0 -50 S2 0 0 T3 0 -50

3 T1 48 -50 S1 7 +10 T2 48 +20 S2 0 0 T3 0 +20

Animal/Human Tissue

45

Wilson M.T. et al. (1998) Vision Research Laboratory and Department of Cellular Biology at the University of Georgia, Athens, GA 30602, USA

Morphological study Specimen: Frog (retina)

Fix.: HPF

Step

Reagent Time [h]

Temp. [°C]

Slope [°Ch-1]


0.1% tannic acid 48 -80

2 2 -20 3 2 +4 4 Wash 3 x in acetone RT 5 Embedding in increasing concentrations

of Epon 812 2 RT

6 Epon 812 100% ON RT 7 Polymerisation +60



Temp. [°C]

Slope [°Ch-1]


0.1% tannic acid 48 -80

S1 12 +5 T2 2 -20 S2 4.8 +5

1

T3 2 +4 2 T1 Wash in acetone (3 x 10 min) 2 +20 30% Epon 812 in acetone (0.5h) 70% Epon 812 in acetone (0.5h) 100% Epon 812 (0.5h) 100% Epon 812 with accelerator S1 2 +20 T2 Polymerisation 48 +60

Animal/Human Tissue

46

Input:

Step Time [h]

Temp. [°C]

Slope [°Ch-1]

T1 48 -80 S1 12 +5 T2 2 -20 S2 4.8 +5

1

T3 2 +4 2 T1 2 +20

S1 2 +20 T2 48 +60 S2 0 0 T3 0 +60

Moreira, J. E. et al. (1998) Departmento de Morfologia Facultade de Medicina (FMRP-USP) 14049-900 Ribeirao Preto Sao Paulo, Brasil


Specimen: Aplysia (muscle), squid giant axons

Fix.: Impact freezing Alternative 1: Immunocytochemistry


Temp. [°C]

1 Acetone containing 0.1% uranyl acetate 24 -80 2 Rinse 2 x in acetone (10 min) -60 3 Rinse 1 x in methanol -60 4 K11M in acetone 50% -60 5 K11M in acetone 75% -60 6 K11M 100% -60 7 K11M 100% -60 8 Polymerisation under UV 300 nm 12 -60 9 Polymerisation under UV 300 nm 12 -40

10 Polymerisation under UV 300 nm 12 -10 11 Polymerisation under UV 300 nm 12 RT 12 Place samples under vacuum ON

Animal/Human Tissue

47

Alternative 2: Morphological study Step Reagent Time

[h] Temp.

[°C]

1 Acetone containing 4% OsO4 36 -80 2 passively warming to -20 3 1 0 4 Acetone containing 1% uranyl acetate 5 Rinse briefly in methanol 6 Substitute with propylene oxide 7 Embedding in Araldite

Our proposal for the AFS: Alternative 1: Immunocytochemistry


Temp. [°C]

Slope [°Ch-1]

1 T1 Acetone containing 0.1% uranyl acetate 24 -80 S1 4 +5 T2 Rinse 2 x in acetone (10 min)

Rinse 1 x in methanol 32.1 -60

K11M in acetone 50% (8h) K11M in acetone 75% (8h) K11M 100% (8h)

K11M 100% (8h) T1 Polymerisation under UV 300 nm 12 -60 S1 1 +20 T2 Polymerisation under UV 300 nm 12 -40 S2 1 +30

!2

T3 Polymerisation under UV 300 nm 12 -10 !3 T1 Polymerisation under UV 300 nm 12 +20

Input:

Step Time [h]

Temp. [°C]

Slope [°Ch-1]

T1 24 -80 S1 4 +5 T2 32.1 -60 S2 0 0

1

T3 0 -60 !2 T1 12 -60

S1 1 +20 T2 12 -40 S2 1 +30 T3 12 -10

!3 T1 12 +20 S1 0 0 T2 0 +20 S2 0 0 T3 0 +20

Animal/Human Tissue

48

Alternative 2: Morphological study Prog. Step Reagent Time

[h] Temp.



1

T3 1 0 T1 Acetone containing 1% uranyl acetate (8h) 9 +20

Rinse briefly in methanol Substitute with propylene oxide (3 x 20 min)

2

Embedding in Araldite Input:

Step Time [h]

Temp. [°C]

Slope [°Ch-1]

T1 36 -80 S1 6 +5 T2 1 -20 S2 4 +5

1

T3 1 0 2 T1 9 +20

S1 0 0 T2 0 +20 S2 0 0 T3 0 +20

Takizawa, T. (1998) Department of anatomy Jichi Medical School 331 Yakushji Minamikawachimachi Tochigi 329-0498, Japan

Immunocytochemistry Specimen: human (hair)


Step Reagent Time

[h] Temp.

[°C]

1 Acetone 72 -80 2 1 -20 3 +4 4 Substitute acetone against dimethylformamide (DMF)

and then against DMF/LR White resin mixtures

5 Exchange 3 x LR White 6 Polymerisation under UV*

Alternatively: oven* +4

+50 to +60

* according to Yokota & Okada (1997)

Animal/Human Tissue

49


[h] Temp.


T1 Acetone 72 -80 S1 6 +5 T2 1 -20 S2 4.8 +5

1

T3 1 +4 T1 Substitute acetone against dimethyl-

formamide (DMF) and then against DMF/LR White resin mixtures (8h)

8 +4

Exchange 3 x LR White

2

Polymerisation under UV* Alternatively: oven*

+4 +50 to

+60

Input:

Step Time [h]

Temp. [°C]

Slope [°Ch-1]

T1 72 -80 S1 6 +5 T2 1 -20 S2 4.8 +5

1

T3 1 +4 2 T1 8 +4

S1 0 0 T2 0 +4 S2 0 0 T3 0 +4

Animal/Human Tissue

50

Gilbert, C. S. & Parmley, R. T. (1998) Carolinas Medical Center Department of Pediatric Research Charlotte North Carolina 28203, USA

Morphological study Specimen: human (neutrophils)


Step Reagent Time

[h] Temp.


1 Acetone containing 4% OsO4 48 -80 2 2 +20 3 -40 4 1 +10 5 -30 6 1 -20 7 1 +4 8 1 RT 9 Wash 3 x in acetone (15 min each) RT

10 Wash 3 x in propylen oxid (20 min each) RT 11 Spurr:propylene oxid 1:1 2 RT 12 Spurr:propylene oxid 2:1 ON RT 13 Spurr 100% 4 RT 14 Polymerisation in fresh Spurr 100%



Temp. [°C]

Slope [°Ch-1]

T1 Acetone containing 4% OsO4 48 -80 S1 2 +20 T2 -40 S2 1 +10

1

T3 -30 T1 1 -20 S1 4.8 +5 T2 1 +4 S2 3.2 +5

2

T3 (1h) 21 +20 Wash 3 x in acetone (15 min each) Wash 3 x in propylen oxid (20 min each) Spurr:propylen oxid 1:1 (2h) Spurr:propylen oxid 2:1 (12h)

Spurr 100% (4h) Polymerisation in fresh Spurr 100%

according to the manufacturers manual

Animal/Human Tissue

51

Input:

Step Time [h]

Temp. [°C]

Slope [°Ch-1]

T1 48 -80 S1 2 +20 T2 -40 S2 1 +10

1

T3 -30 2 T1 1 -20

S1 4.8 +5 T2 1 +4 S2 3.2 +5 T3 21 +20

Nusser, Z. et al. (1995) Institute of Experimental Medicine Hungarian Academy of Sciences H 1450 Budapest POB 67, Hungary

Immunocytochemistry Specimen: brain (cat, rat)

Fix.: Chem. fix, followed by cryoprotection and impact freezing

Step Reagent Time

[h] Temp.


1 Methanol* 36 -80 2 +10 3 HM20:methanol 1:1 1.5 -50 4 HM20:methanol 2:1 1.5 -50 5 pure HM20 1.5 -50 6 pure HM20 ON -50 7 fresh HM20 -50 8 Polymerisation under UV 48 -50

*FS and embedding/polymerisation according to Baude et al. 1993 Our proposal for the AFS:


Temp. [°C]

Slope [°Ch-1]

T1 Methanol 36 -80 S1 3 +10 T2 HM20:methanol 1:1 (1.5h) 16.5 -50

HM20:methanol 2:1 (1.5h) -50

1

pure HM20 (1.5h) -50 pure HM20 (12h) -50 fresh HM20 -50

!2 T1 Polymerisation under UV 48 -50

Animal/Human Tissue

52

Input: Step Time

[h] Temp.


T1 36 -80 S1 3 +10 T2 16.5 -50 S2 0 0

1

T3 0 -50 2 T1 48 -50

S1 0 0 T2 0 -50 S2 0 0 T3 0 -50

Studer et al. (1995) Maurice E. Mueller Institute for Biomechanics University of Bern P.O. Box 30 3010 Bern, Switzerland

Morphological study Specimen: bovine humeral head articular

cartilage Fix.: HPF

Step Reagent Time

[h] Temp.

[°C]

1 Acetone containing 2% OsO4 17 -90 2 12 -60 3 12 -30 4 1 0 5 Wash 3 x in acetone 0 6 Epon 812 30% 3 6 Epon 812 70% 72 6 Epon 812 100% 72 7 Polymerisation 120 +60

Animal/Human Tissue

53



Temp. [°C]

Slope [°Ch-1]


1

T3 12 -30 T1 (1h) 1.5 0

Wash in acetone (3 x 10 min) S1 1 +20

2

T2 Epon 812 30% (3h) 175 +20 Epon 812 70% (72h) Epon 812 100% (72h)

S2 1 +40

T3 Polymerisation 120 +60 Input:

Step Time [h]

Temp. [°C]

Slope [°Ch-1]

T1 17 -90 S1 6 +5 T2 12 -60 S2 6 +5

1

T3 12 -30 2 T1 1.5 0

S1 1 +20 T2 175 +20 S2 1 +40 T3 120 +60

Eggli P.S. & Graber W. (1994) Institute of Anatomy University of Bern CH-3000 Bern, Switzerland

Morphological study Specimen: Rat (ciliary body)


Step Reagent Time

[h] Temp.

[°C]

1 Acetone containing 2% OsO4 8 -90 2 8 -60 3 8 -30 4 Acetone +4 5 Embedding in Epon 812 RT 6 Polymerisation +60

Animal/Human Tissue

54



Temp. [°C]

Slope [°Ch-1]


1

T3 8 -30 2 T1 8 +4 S1 2 +8 T2 Epon:acetone 1:2 (4h) 20 +20 Epon:acetone 1:1 (1h) Epon:acetone 2:1 (1h) Epon:acetone 2:1 (1h) Epon 100% (1h) Epon containing accelerator (12h) S2 1 +40 T3 Polymerisation 48 +60

Input:

Step Time [h]

Temp. [°C]

Slope [°Ch-1]

T1 8 -90 S1 6 +5 T2 8 -60 S2 6 +5

1

T3 8 -30 2 T1 8 +4

S1 2 +8 T2 20 +20 S2 1 +40 T3 48 +60

Animal/Human Tissue

55

Keene D.R. & McDonald K.(1993) Portland Shrine Research Unit 3101 SW Sam Jackson Pk. Rd. Portland, OR, 97201 USA

Morphological study Specimen: Chick embryos (Sternal cartilage) Human (Foreskin, costal

cartilage) Fix.: HPF

Step Reagent Time

[h] Temp.


1 Acetone containing 1% OsO4 72 -90 2 6-8 +11.25

to +15 3 Rinse in acetone 0 4 Embedding in Epon-Araldite RT 5 Polymerisation 48 +60 6



Temp. [°C]

Slope [°Ch-1]

T1 Acetone containing 1% OsO4 72 -90 S1 7.8 +11.5 T2 Rinse in acetone 1 0 S2 4 +5

1

T3 Epon-Araldite:acetone 1:2 (4h) 20 +20 Epon-Araldite:acetone 1:1 (1h) Epon-Araldite:acetone 2:1 (1h) Epon-Araldite:acetone 2:1 (1h) Epon-Araldite 100% (1h) Epon-Araldite containing

accelerator (12h)


Step Time [h]

Temp. [°C]

Slope [°Ch-1]

T1 72 -90 S1 7.8 +11.5 T2 1 0 S2 4 +5

1

T3 20 +20 2 T1 48 +60

S1 0 0 T2 0 +60 S2 0 0 T3 0 +60

Viruses, Bacteria, Cells

56

Viruses, Bacteria, Cells Ellis, S. et al. (2000) Peter MacCallum Cancer Institute Melbourne Victoria, Australia

Immunocytochemistry Specimen: Chinese hamster ovary cells

Fix.: Chem. fix, followed by cryoprotection and impact freezing

Step Reagent Time

[h] Temp.


1 Methanol 96 – 120 -91 2 Warm to -50 3 Embedding in HM20 -50 4 Polymerisation under UV -50



Temp. [°C]

Slope [°Ch-1]

T1 Methanol 96 -91 S1 10 +4 T2 Wash in methanol 4 x 15 min 8 -50

Methanol:HM20 3:1 (1h)

1

Methanol:HM20 2:1 (1h) Methanol:HM20 1:1 (1h) pure HM20 (4h)


Step Time [h]

Temp. [°C]

Slope [°Ch-1]

T1 96 -91 S1 10 +4 T2 8 -50 S2 0 0

1

T3 0 -50 !2 T1 36 -50

S1 0 0 T2 0 -50 S2 0 0 T3 0 -50


57

Horstmann, H. (pers. com.) Membrane Biology Laboratory Institute of Molecular and Cell Biology 30 Medical Drive Singapore 117609, Singapure

Morphological study Specimen: Cell monolayer


Alternative 1: HM20 Monostep


Temp. [°C]

Slope [°Ch-1]

1 Methanol containing 0.5% uranyl acetate 12 -85 2 9 +5 3 0.5 10.5 -40 4 3 -40 5 Methanol:HM20 3:1 (1h) 6 Methanol:HM20 2:1 (1h) 7 Methanol:HM20 1:1 (1h) 8 pure HM20 (4h) 9 Polymerisation under UV 36 -40

Alternative 2: epoxy resins


Temp. [°C]

Slope [°Ch-1]

1 Acetone containing 0.5% uranyl acetate,

2% OsO4*

12 -90

2 6 +5 3 2 -60 4 2 +20 5 2 -20 6 0.5 +4 7 Embedding in Epon 812, Spurr, Araldite

• Alternatively: Acetone containing 0.5% uranyl acetate, 2% OsO4, 0.25% GAD Our proposal for the AFS: Alternative 1


Temp. [°C]

Slope [°Ch-1]

T1 Methanol containing 0.5% uranyl acetate 12 -85 S1 9 +5 T2 (0.5h) 10.5 -40

1

(3h) -40 Methanol:HM20 3:1 (1h) Methanol:HM20 2:1 (1h) Methanol:HM20 1:1 (1h) pure HM20 (4h)

!2 T1 Polymerisation under UV 36 -40


58

Input: Step Time

[h] Temp.


T1 12 -85 S1 9 +5 T2 10.5 -40 S2 0 0

1

T3 0 -40 !2 T1 36 -40

S1 0 0 T2 0 -40 S2 0 0 T3 0 -40

Alternative 2


Temp. [°C]

Slope [°Ch-1]

T1 Acetone containing 0.5% uranyl acetate,

2% OsO4*

12 -90

S1 6 +5 T2 2 -60

1

S2 2 +20 T3 2 -20

2 T1 0.5 +4 Embedding in Epon 812, Spurr, Araldite

Input:

Step Time [h]

Temp. [°C]

Slope [°Ch-1]

T1 12 -90 S1 6 +5 T2 2 -60 S2 2 +20

1

T3 2 -20 2 T1 0.5 +4

S1 0 0 T2 0 +4 S2 0 0 T3 0 +4


59

Holland D.J. et al. (1998) Center for Virus Research Westmead Institutes of Health Research Westmead Hospital & the University of Sidney, Westmead, Sidney NSW 2145, Australia

Immunocytochemistry Specimen: Virus (Herpes simplex)

in cell culture Fix.: chemical


Temp. [°C]

Slope [°Ch-1]

1 Methanol 40 -90 2 “ 9 +5 3 Rinse in methanol -45 4 Methanol:HM20 1:1 2 -45 5 Methanol:HM20 1:2 2 -45 6 HM20 pure ON -45 7 Polymerisation under UV 48 -45



Temp. [°C]

Slope [°Ch-1]

T1 Methanol 40 -90 S1 9 +5 T2 Rinse in methanol 16 -45

1

Methanol:HM20 1:1 (2h) Methanol:HM20 1:2 (2h) HM20 pure (12h)


Step Time [h]

Temp. [°C]

Slope [°Ch-1]

T1 40 -90 S1 9 +5 T2 16 -45 S2 0 0

1

T3 0 -45 !2 T1 48 -45

S1 0 0 T2 0 -45 S2 0 0 T3 0 -45


60

Neuhaus E.M. et al. (1998) Departmentof Molecular Cell Research Max-Planck-Institute for Medical Research Jahnstr. 29 D 69120 Heidelberg, Germany

Immunocytochemistry, morphological study

Specimen: Dictyostelium discoideum Fix.: plunge freezing on sapphire discs



Temp. [°C]

Slope [°Ch-1]

1 Methanol containing 1.5% uranyl acetate 36 -85 2 8 +5 3 Wash 3 x in methanol -45 4 Methanol:HM20 2:1 1 -45 5 Methanol:HM20 2:1 1 -45 6 Methanol:HM20 1:2 1 -45 7 Methanol:HM20 1:2 1 -45 8 pure HM20 2 -45 9 Polymerisation under UV 36 -45

10 RT Alternative 2: Morphological study


Temp. [°C]

Slope [°Ch-1]

1 Methanol containing 1.5% uranyl acetate 36 -85 2 8 +5 3 Acetone containing 1% OsO4 2 -45 4 pure acetone -30 5 Acetone:Epon 1:1 RT 6 Polymerisation 48 +56

Our proposal for the AFS: Alternative 1: Immunocytochemistry


Temp. [°C]

Slope [°Ch-1]

T1 Methanol containing 1.5% uranyl acetate 36 -85 S1 8 +5 T2 Wash 3 x in methanol 6.1 -45

1

Methanol:HM20 2:1 (1h) Methanol:HM20 2:1 (1h) Methanol:HM20 1:2 (1h) Methanol:HM20 1:2 (1h) pure HM20 (2h)

!2 T1 Polymerisation under UV 36 -45 S1 4.3 +15 T2 0 +20


61

Input: Step Time

[h] Temp.


T1 36 -85 S1 8 +5 T2 6.1 -45 S2 0 0

1

T3 0 -45 !2 T1 36 -45

S1 4.3 +15 T2 0 +20 S2 0 0 T3 0 +20

Alternative 2:


Temp. [°C]

Slope [°Ch-1]

T1 Methanol containing 1.5% uranyl acetate 36 -85 S1 8 +5 T2 Acetone containing 1% OsO4 2 -45

1

S2 3 +5 T3 pure acetone (2h) 6 -30 Epon:acetone 1:1 (2h) pure Epon (2h)


Step Time [h]

Temp. [°C]

Slope [°Ch-1]

T1 36 -85 S1 8 +5 T2 2 -45 S2 3 +5

1

T3 6 -30 2 T1 48 +56

S1 0 0 T2 0 +56 S2 0 0 T3 0 +56


62

Wild, P. et al. (1998) Institute of Veterinary Anatomy Laboratory for Electron Microscopy Winterthurerstr. 260 CH-8057 Zürich, Switzerland

Morphological study Specimen: Virus (BHV 1, BHV 5)

Fix.: plunge freezing

Step Reagent Time

[h] Temp.


1 Acetone containing 0.25% GAD,

0.5% OsO4 4 -90

2 18 +5 3 1 0 4 briefly wash with pure acetone +4 5 Embedding in Epon 6 +4 6 Polymerisation 48 +60



Temp. [°C]

Slope [°Ch-1]

T1 Acetone containing 0.25% GAD,

0.5% OsO4 4 -90

S1 18 +5 T2 1 0 S2 1 +4 T3 briefly wash with pure acetone 6 +4

1

Epon:acetone 1:2 (1h) Epon:acetone 1:1 (1h) Epon:acetone 2:1 (2h) Epon100% (2h)


Step Time [h]

Temp. [°C]

Slope [°Ch-1]

T1 4 -90 S1 18 +5 T2 1 0 S2 1 +4

1

T3 6 +4 2 T1 48 +60

S1 0 0 T2 0 +60 S2 0 0 T3 0 +60


63

Erk I. et al. (1998) Centre de Genetique Moleculaire CNRS Avenue de la Terasse F-91190 Gif sur Yvette, France

Morphological study Specimen: bacteria, yeast, paramecium

Fix.: HPF, plunge freezing

Step

Reagent Time

[h] Temp.


1 Acetone containing 5% OsO4 * 72 -85 2 3 +18.33 3 1 -30 4 Wash in acetone RT 5 Embedding in Epon

* in some cases 0.2% tannic acid added Erk et al. (1998): “The substitution time (three days) can be adapted for different tissues. We found that shorter periods led to poorer results; the samples were not perfectly embedded in the plastic resin. For the samples studied in this paper, we did not find any improvement for times longer than 3 days.” Our proposal for the Leica AFS:


Temp. [°C]

Slope [°Ch-1]

T1 Acetone containing 5% OsO4 72 -85 S1 3 +18.5 T2 1 -30 S2 5 +10

1

T3 Wash in acetone 35 +20 Epon:acetone 1:2 (3h) Epon:acetone 2:1 (12h) pure Epon (8h) pure Epon (12h)


Step Time [h]

Temp. [°C]

Slope [°Ch-1]

T1 72 -85 S1 3 +18.5 T2 1 -30 S2 5 +10

1

T3 35 +20 2 T1 72 +60

S1 0 0 T2 0 +60 S2 0 0 T3 0 +60


64

Roch A.M. et al. (1997) Lab. d'Immunochimie Fac. Med. Lyon-Sud Chemin du Petit Revoyet BP 12 F-69921 Oullins Cedex , France

Immunocytochemistry Specimen: HeLa cells Fix.: Impact Freezing

Step Reagent Time

[h] Temp.


1 Acetone* 72 -90 2 6 +10 3 2 -30 4 1 RT 5 Wash in acetone** 6 Embedding in Epon** 7 Polymerisation 24 +55

* alternatively: acetone containing 5% OsO4 ** alternatively: wash in ethanol, embedding in LR White Our proposal for the AFS:


Temp. [°C]

Slope [°Ch-1]

T1 Acetone 72 -90 S1 6 +10 T2 2 -30 S2 10 +5

1

T3 (1h) 36 +20 Wash in acetone Epon:acetone 1:2 (3h) Epon:acetone 2:1 (12h) Pure Epon (8h) Pure Epon (12h)


Step Time [h]

Temp. [°C]

Slope [°Ch-1]

T1 72 -90 S1 6 +10 T2 2 -30 S2 10 +5

1

T3 36 +20 2 T1 24 +55

S1 0 0 T2 0 +55 S2 0 0 T3 0 +55


65

Graham, L., Beveridge, T.J. (1990) Department of Microbiology College of Biological Sciences University of Guelph Ontario, Canada N1G 2W1

Morphological study Specimen: E. coli, Bacillus subtilis


Step Reagent Time

[h] Temp.



and 2% uranyl acetate 72 -80

2 RT 3 Wash in fresh acetone 6 x 15 min 4 Epon 812: acetone 1:1 ON RT 5 Fresh Epon 812 6 Polymerisation 36 +60



Temp. [°C]

Slope [°Ch-1]


and 2% uranyl acetate 72 -80

S1 10 +10

1

T2 Wash in fresh acetone 6 x 15 min 13.5 +20 Epon 812: acetone 1:1 (12h) Fresh Epon 812 S2 1 +40 T3 Polymerisation 36 +60

Input:

Step Time [h]

Temp. [°C]

Slope [°Ch-1]

T1 72 -80 S1 10 +10 T2 13.5 +20 S2 1 +40

1

T3 36 +60

PLT for TEM

66

PLT for TEM PLT according to Ludwig Edelmann (personal communication) Ludwig Edelmann Medizinische Biologie, Theoretische Medizin Universität des Saarlandes D 66421 Homburg/Saar Germany L.E.`s proposal for the AFS:


Temp. [°C]

Slope [°Ch-1]

T1 EtOH 30% 0.5 0 S1 EtOH 50% 1 -20 T2 EtOH 75% 0.2 -20 S2 1 -10

1

T3 EtOH 100% 1 -30 T1 EtOH 100% 1 -30 S1 0 0 T2 EtOH:Lowicryl 1:1 1 -30 S2 0 0

2

T3 EtOH:Lowicryl 1:2 1 -30 T1 Lowicryl pure 100% 1 -30 S1 0 0 T2 Lowicryl pure 100% ON -30 S2 0 0

3

!T3 Polymerisation under UV 48 -30 Input:

Step Time [h]

Temp. [°C]

Slope [°Ch-1]

T1 0.5 0 S1 1 -20 T2 0.2 -20 S2 1 -10

1

T3 1 -30 2 T1 1 -30

S1 0 0 T2 1 -30 S2 0 0 T3 1 -30

3 T1 1 -30 S1 0 0 T2 12 -30 S2 0 0 !T3 48 -30

PLT for TEM

67

Gounon P. & Rolland J.P. (1998) Institute Pasteur Station Central de Microscopie Electronique 25, rue du Docteur Roux 75724 Paris cedex 15, France

Immunocytochemistry Specimen: Mouse (pancreas)

Fix.: Chem.

Alternative 1:


Temp. [°C]

Slope [°Ch-1]

1 Ethanol 50% 0.5 +4 2 Ethanol 70% 0.5 +4 3 Ethanol 90% 0.5 +4 4 Ethanol 100% 0.5 +4 5 Ethanol 100% 0.5 +4 6 2.4 -10 7 -20 8 Unicryl:Ethanol 1:2 0.5 -20 9 Unicryl:Ethanol 2 :1 0.5 -20

10 Unicryl 1 -20 11 Unicryl ON -20 12 Polymerisation under UV 96 -20 13 Curing 168 +4

Alternative 2:


Temp. [°C]

Slope [°Ch-1]

1 Ethanol 50% 0.5 +4 2 Ethanol 70% 0.5 +4 3 Ethanol 90% 0.5 +4 4 Ethanol 100% 0.5 +4 5 Ethanol 100% 0.5 +4 6 3.9 -10 7 Unicryl:Ethanol 1:2 0.5 -35 8 Unicryl:Ethanol 2 :1 0.5 -35 9 Unicryl* 1 -35

10 Unicryl* ON -35 11 Polymerisation under UV < 24 -35 12 Curing 48 -35 13 3.7 +15 14 RT

* 0.05% benzoidin ethyl ether was added to Unicryl

PLT for TEM

68

Our proposal for the AFS: Alternative 1:


Temp. [°C]

Slope [°Ch-1]

T1 Ethanol 50% (0.5h) 2.5 +4

Ethanol 70% (0.5h) Ethanol 90% (0.5h) Ethanol 100% (0.5h) Ethanol 100% (0.5h)

S1 2.4 -10 T2 Unicryl:Ethanol 1:2 (0.5h) 14 -20

Unicryl:Ethanol 2 :1 (0.5h) Unicryl (1h)

1

Unicryl (12h) !2 T1 Polymerisation under UV 96 -20

S2 4.8 +5 T2 Curing 168 +4

Input:

Step Time [h]

Temp. [°C]

Slope [°Ch-1]

T1 2.5 +4 S1 2.4 -10 T2 14 -20 S2 0 0

1

T3 0 -20 !2 T1 96 -20

S1 4.8 +5 T2 168 +4 S2 0 0 T3 0 +4

Alternative 2: Prog. Step Reagent Time

[h] Temp.


T1 Ethanol 50% (0.5h) 2.5 +4

Ethanol 70% (0.5h) Ethanol 90% (0.5h) Ethanol 100% (0.5h) Ethanol 100% (0.5h)

S1 3.9 -10 T2 Unicryl:Ethanol 1:2 (0.5h) 14 -35

Unicryl:Ethanol 2:1(0.5h) Unicryl (1h)

1

Unicryl (12h) !2 T1 Polymerisation under UV (30h) 78 -35

Curing (48) S1 3.7 +15 T2 1 +20

PLT for TEM

69

Input: Step Time

[h] Temp.


T1 2.5 +4 S1 3.9 -10 T2 14 -35 S2 0 0

1

T3 0 -35 !2 T1 78 -35

S1 3.7 +15 T2 1 +20 S2 0 0 T3 0 +20

Whitehouse et al. (1984) Unite de Microscopie Electronique et de Genetique Moleculaire Departement de Biologie Moleculaire Institut Pasteur 25 rue du Docteur Roux 75724 Paris Cedex 15, France

Immunocytochemistry Specimen: Bacteriophage l in E. coli

Fix.: Chem.

Step Reagent Time

[h] Temp.


1 Ethanol 25% 0.5 0 2 Ethanol 50 % 2 – 3 min 400 3 1 -20 4 Ethanol 75% 1 -20 5 Ethanol 90% 2 – 3 min 100 6 1 -25 7 Ethanol 100 % 1 -25 8 Lowicryl:Ethanol 1:1 1 -25 9 Lowicryl:Ethanol 2:1 1 -25

10 Lowicryl 1 -25 11 Lowicryl ON -20 12 Polymerisation under UV 6 - 10 -18 13 Polymerisation under UV 34 - 38 +4 14 Curing 72 - 144

PLT for TEM

70


[h] Temp.


T1 Ethanol 25%, 0.5 0 S1 Ethanol 50% 1 -20 T2 Ethanol 75% 1 -20 S2 Ethanol 90% 1 -5

1

T3 Ethanol 100% 1 -25 2 T1 Lowicryl:Ethanol 1:1 (1h) 3 -25 Lowicryl:Ethanol 2:1 (1h) Lowicryl (1h) S1 1 +5 T2 Lowicryl pure 12 -20

!3 T1 Polymerisation under UV (24h) 48 +4 Polymerisation under UV (24h) S1 3.2 +5 T2 Curing outside AFS 72 +20

Input:

Step Time [h]

Temp. [°C]

Slope [°Ch-1]

T1 0.5 0 S1 1 -20 T2 1 -20 S2 1 -5

1

T3 1 -25 2 T1 3 -25

S1 1 +5 T2 12 -20 S2 0 0 T3 0 -20

!3 T1 48 +4 S1 3.2 +5 T2 72 +20 S2 0 0 T3 0 +20

LTE for X-ray microanalysis

71

LTE for X-ray microanalysis Thirion S. et al. (1997) Laboratoire de Physiologie Cellulaire et Moleculaire CNRS URA 1938 Nice, France

X-ray microanalysis Specimen: rat (nerve endings)


Step

Reagent Time

[h] Temp.


1 Ethanol containing 20 mM oxalic acid 72 -120 -88 2 49 - 51 +2.1 to

+2.2 3 +20 4 Ethanol:Epon 1:1 2 +20 5 Embedding in Epon



Temp. [°C]

Slope [°Ch-1]

T1 Ethanol containing

20 mM oxalic acid 120 -88

S1 50 +2 T2 Epon:Ethanoll 1:1 (2h) 14 +20

pure Epon (12h)

1

Pure Epon + 1.5% BDMA S2 1 +40 T3 Polymerisation 72 +60

Input:

Step Time [h]

Temp. [°C]

Slope [°Ch-1]

T1 120 -88 S1 50 +2 T2 14 +20 S2 1 +40

1

T3 72 +60


72

Bordat, C. & Cournot, G. (1998) CNRS URA 583 INRA, LNSA Batiment 221 F-78350 Jouy en Josas, France

Spectroscopic imaging Specimen: rat (fetal bone)

Fix.: HPF

Step Reagent Time

[h] Temp.

[°C]

1 Acetone 72 -90 2 Progressive infiltration

with HM23 72

3 Polymerisation under UV 24 -80 Our proposal for the Leica AFS:


Temp. [°C]

Slope [°Ch-1]

T1 Acetone (72h) 144 -90

Progressive infiltration with HM23 according to the

manufacturers manual (72h)

S1 2 +5

1

T2 Polymerisation under UV 24 -80 Input:

Step Time [h]

Temp. [°C]

Slope [°Ch-1]

T1 144 -90 S1 2 +5 T2 24 -80 S2 0 0

1

T3 0 -80


73

Orlovich D.A. & Ashford A.E. (1995) School of Biological Science The University of New South Wales NSW Sydney 2052, Australia

X-ray microanalysis Specimen: salt/dextran droplets

Fix.: impact freezing

Step

Reagent Time

[h] Temp.

[°C]

1 Diethyl ether* 504 -85 2 24 -20 3 2 +4 4 2 +20 5 Embedding in modified Spurr 72 - 120 RT 6 Fresh modified Spurr 7 Polymerisation 24 +70 8 Polymerisation 168 RT

* alternatively: Diethyl ether containing 20% acrolein. Our proposal for the AFS:


Temp. [°C]

Slope [°Ch-1]

T1 Diethyl ether* 504 -85 S1 6.5 +10 T2 24 -20 S2 4.8 +5

1

T3 2 +4 2 T1 (2h) 122 +20 Embedding in modified Spurr (120h) Fresh modified Spurr S1 2 +25 T2 Polymerisation 24 +70 S2 2 -25 T3 Polymerisation 168 +20


74

Input: Step Time

[h] Temp.


T1 504 -85 S1 6.5 +10 T2 24 -20 S2 4.8 +5

1

T3 2 +4 2 T1 122 +20

S1 2 +25 T2 24 +70 S2 2 -25 T3 168 +20

SEM and LM Ap Gwynn, I. et al. (2000) Institute of Biological Sciences The University of Wales Aberystwyth, Wales SY23 3DA, UK

Morphological study, SEM Specimen: Rabbit (cartilage)


Alternative 1:


Temp. [°C]

Slope [°Ch-1]

1 Acetone*, daily change 192 -80 2 20 +5 3 Critical point dry, sputter coat +20

* alternatively: acetone containing 2% acrolein Alternative 2:


Temp. [°C]

Slope [°Ch-1]

1 Methanol:acetone 1:2 containing 10% acrolein, 0.2% tannic acid

168 -80

2 Rinse in acetone (20 min) 3 Methanol:acetone 1:9

containing 5% GAD 96 -23

4 8.6 +5 5 Critical point dry, sputter coat +20

Our proposal for the AFS: Alternative 1:


Temp. [°C]

Slope [°Ch-1]

T1 Acetone, daily change 192 -80 S1 20 +5

1

Critical point dry, sputter coat +20

SEM and LM

75

Input:

Step Time [h]

Temp. [°C]

Slope [°Ch-1]

T1 192 -80 S1 20 +5 T2 0 +20 S2 0 0

1

T3 0 +20 Alternative 2:


Temp. [°C]

Slope [°Ch-1]

T1 Methanol:acetone 1:2 containing

10% acrolein, 0.2% tannic acid, rinse in acetone (20 min)

168 -80

S1 11.4 +5

1

T2 Methanol:acetone 1:9 containing 5% GAD

96 -23

S2 8.6 +5 T3 1 +20 Critical point dry, sputter coat

Input:

Step Time [h]

Temp. [°C]

Slope [°Ch-1]

T1 168 -80 S1 11.4 +5 T2 96 -23 S2 8.6 +5

1

T3 1 +20

SEM and LM

76

He, Y. & Wetzstein, H.Y. (1997) Department of Horticulture University of Georgia, Athens Georgia USA

Immunofluorescence, LM Specimen: Nicotiana (protoplasts),

Tradescantia (pollen) Mouse (myeloma)



Temp. [°C]

1 Methanol containing 2% form-

aldehyde, 0.01% glutaraldehyde 48 -80

2 6 -20 3 6 +4 4 Immunofluorescence RT



Temp. [°C]

Slope [°Ch-1]

T1 Methanol containing 2% form-

aldehyde, 0.01% glutaraldehyde 48 -80

S1 12 +5

1

T2 6 -20 S2 4.8 +5 T3 6 +4

Input:

Step Time [h]

Temp. [°C]

Slope [°Ch-1]

T1 48 -80 S1 12 +5 T2 6 -20 S2 4.8 +5

1

T3 6 +4 He, Y. & Wetzstein, H.Y. (1997): “The present study uses lower concentrations of glutaraldehyde (0.01%) in combination with formaldehyde (2%) which produced excellent labelling with low background. Moreover, our results show that substitution using methanol is superior to acetone […]”

References

77

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