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  • 7/27/2019 Only 1/50 ml (20 l) of Water-soluble Aminoplastic Resin Is Enough to Embed One Biological Sample for EM Study

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    Only 1/50 ml (20 l) of Water-soluble Aminoplastic

    Resin Is Enough to Embed One Biological Sample for EM

    Study of Membrane and Lipid Ultrastructure

    Johnson GaoRetired professor in Cell Biology

    8/8/2013

    e-mail: [email protected]

    It is a shame that the lipid ultra-structural research is far less developed

    than the ultra-structural researches ofprotein, polysaccharide, and nucleic

    acid based subcellular compartments,although all of those four substances are

    the common structural buildingmaterials and they almost share equal

    importance to the existence of livingthings. The delay in lipid ultra-structural

    research is mainly due to the lack of aproper water-soluble embedding

    medium commercially available on the

    market that can avoid organic solventsfor dehydrating and infiltration agent,which will extract fat and lipid.

    If someone tells you that only

    1/50 ml (20 l) of water-soluble resin isenough to run the infiltration and

    embedding of biological sample in onesingle step for electron microscopic

    study of ultrastructure of lipid, you may

    say: Are you teasing? Is it reallypossible that so little amount of

    embedding medium can do the job?

    Or, you may say: It is fantastic. It issuper saving. Tell me how to do with

    that kind of embedding, immediately.Tell you the truth. The technique had

    existed for long time, which was in theyear 1982. It was called as silylated

    aminoplastic embedding method.[1] Ithad nicely shown good result in the

    study of lipovitalline molecule changesin the first cleavage of frog. And, with

    that method, I had published the worldsfirst batch of color electron

    micrograph.[2] The aim of my work atthat time was that I wanted to join a

    fundamental discussion - If the humanbeing was created by the God, or,

    through the long course of evolution?

    and I heard that Friedrich Engels had afamous saying about life and chemistry(1894). His definition reads: Life is the

    existence form of proteic structures, andthis existence form consists essentially

    in the constant self-renewal of thechemical components of these

    structures. Unfortunately, thepublication of my research was written

    in Chinese. So, my method was not

    known by most of American scientists.Recently, to reduce the difficulty caused

    by language barrier, I had described that

    method in English in a mini review. [3]

    In that small pamphlet, four main steps

    of silylated aminoplastic embeddingmethod are dually presented.

    mailto:[email protected]:[email protected]

  • 7/27/2019 Only 1/50 ml (20 l) of Water-soluble Aminoplastic Resin Is Enough to Embed One Biological Sample for EM Study

    2/3

    Since that aminoplastic resin is

    not commercialized yet, if you want totry it and to verify whether 20 l of

    water-soluble aminoplastic is really

    enough to embed you sample for EMstudy of lipid with your own project,

    you have to synthesis the resin byyourself.

    Here is the operation instruction

    for you. First, dissolve 30 g of

    paraformaldehyde and 10 g of urea in30 ml of 25 % glutaraldehyde in a

    beaker and 7 ml of distilled water is

    added. Adjust the pH to 8.0 by addingfew drops of 25~28% ammonium water.Transfer the mixture into a reflux and

    keep it at 80 C for 3-5 hour until the

    mixture turns clear. Because theaminoplastic will cause self-

    polymerization at low pH condition,

    care must be taken that the reaction inthe first hour may cause a drop of pH to

    6. If you do not use the ammonium

    water (about 50 drops) to bring it backto the weak alkaline condition and

    adjust the pH to 7.5, the whole mixturemay cause polymerization and become

    useless. Second, prepare the working

    medium for infiltration and embedding.

    It is composed of 400 l of aminoplasticsolution, 60 l of PEG-400 and 20 l of

    30% NH4Cl. Adjust the mixture to pH 4

    with HCl. And now 20 l of that

    working medium is enough to do theinfiltration and dehydration for one

    sample, which is performed in a

    combined step in a plastic ring about 2mm inner diameter and 2 mm in height

    at 4 C overnight in a mini desiccator,using P2O5 as an effective dehydrating

    agent. Of course, you must trim yoursample to the size of 1/4 mm3, or, 1/8

    mm3. Polymerization of the resin is

    performed at 37 C for 30 min. Then,cut out the unrelated part of cured resin.

    The sample shall be followed by 60 C

    incubation, for overnight, to increase itscross-linking.[1, 3] You must hook asilylation technique to transform all of

    -OH groups in the solidified resin block

    to the hydrophobic organic siliconresidues[4] that repels water effectively

    and that makes cutting for easy. The

    tools needed for silylating procedure areshown in the diagram 2 in the literature

    cited No. 3. Finally, the sample must be

    mounted on a plastic rod forsectioning.[3] The thin sections can be

    stained with normal EM sectionstaining.

    Anyone who is interested in

    develop a kit of silylated aminoplastic

    embedding may touch me for furtherknow-how knowledge.

  • 7/27/2019 Only 1/50 ml (20 l) of Water-soluble Aminoplastic Resin Is Enough to Embed One Biological Sample for EM Study

    3/3

    Literature cited

    [1]. Gao, K. X. and M. Y. Zhou (1982)Silylated aminoplastic used as a water

    soluble embedding medium for electronmicroscopic study of lipids. Acta

    Biologiae Experimentalis Sinica.,15(1): 57-65.

    [2]. Gao, K. X. and K.Y. Ku (1983)

    Ultrastructural changes of yolk platelets

    during the first cleavage of thefertilized eggs of Rana amurensis. Acta

    Experimentalis Sinica, 16(3): 325-337.

    [3]. Gao, Johnson (2013) A Mini

    Review of Water-soluble Aminoplasticfor Electron Microscopy: The Story

    Behind the World's First Color ElectroMicrograph.

    http://www.lulu.com/shop/johnson-

    gao/a-mini-review-of-water-soluble-aminoplastic-for-electron-microscopy-

    the-story-behind-the-worlds-first-color-electro-micrograph/paperback/product-21127316.html

    [4]. Chambaz, E. M. and E. C. Horning

    (1969) Conversion of steroids totrimethyl derivatives for gas phase

    analytical studies: Reaction of silylatingreagents. Anal. Biochem., 30: 7-24.

    http://www.lulu.com/shop/johnson-gao/a-mini-review-of-water-soluble-aminoplastic-for-electron-microscopy-the-story-behind-the-worlds-first-color-electro-micrograph/paperback/product-21127316.htmlhttp://www.lulu.com/shop/johnson-gao/a-mini-review-of-water-soluble-aminoplastic-for-electron-microscopy-the-story-behind-the-worlds-first-color-electro-micrograph/paperback/product-21127316.htmlhttp://www.lulu.com/shop/johnson-gao/a-mini-review-of-water-soluble-aminoplastic-for-electron-microscopy-the-story-behind-the-worlds-first-color-electro-micrograph/paperback/product-21127316.htmlhttp://www.lulu.com/shop/johnson-gao/a-mini-review-of-water-soluble-aminoplastic-for-electron-microscopy-the-story-behind-the-worlds-first-color-electro-micrograph/paperback/product-21127316.htmlhttp://www.lulu.com/shop/johnson-gao/a-mini-review-of-water-soluble-aminoplastic-for-electron-microscopy-the-story-behind-the-worlds-first-color-electro-micrograph/paperback/product-21127316.htmlhttp://www.lulu.com/shop/johnson-gao/a-mini-review-of-water-soluble-aminoplastic-for-electron-microscopy-the-story-behind-the-worlds-first-color-electro-micrograph/paperback/product-21127316.htmlhttp://www.lulu.com/shop/johnson-gao/a-mini-review-of-water-soluble-aminoplastic-for-electron-microscopy-the-story-behind-the-worlds-first-color-electro-micrograph/paperback/product-21127316.htmlhttp://www.lulu.com/shop/johnson-gao/a-mini-review-of-water-soluble-aminoplastic-for-electron-microscopy-the-story-behind-the-worlds-first-color-electro-micrograph/paperback/product-21127316.htmlhttp://www.lulu.com/shop/johnson-gao/a-mini-review-of-water-soluble-aminoplastic-for-electron-microscopy-the-story-behind-the-worlds-first-color-electro-micrograph/paperback/product-21127316.htmlhttp://www.lulu.com/shop/johnson-gao/a-mini-review-of-water-soluble-aminoplastic-for-electron-microscopy-the-story-behind-the-worlds-first-color-electro-micrograph/paperback/product-21127316.htmlhttp://www.lulu.com/shop/johnson-gao/a-mini-review-of-water-soluble-aminoplastic-for-electron-microscopy-the-story-behind-the-worlds-first-color-electro-micrograph/paperback/product-21127316.htmlhttp://www.lulu.com/shop/johnson-gao/a-mini-review-of-water-soluble-aminoplastic-for-electron-microscopy-the-story-behind-the-worlds-first-color-electro-micrograph/paperback/product-21127316.html

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