pigments and minerals

8/3/2019 Pigments and Minerals

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Chapter 7 |Pigments and MineralsSusieSmith,BS,HTL(ASCP)andLaurieJ.Hafer,PhD

RevisedandupdatedbyJohnKiernanMB,ChB,PhD,DSc

his chapter is concerned with some non-living intracellular inclu-

sions and extracellular deposits, especially those that are commonly

encountered in human histopathology.

Pigments

n the science o colorants, a pigment is an insoluble white or colored

substance that can be suspended in a liquid or application to a

surace (atinpigmentum, paint) or incorporated into a solid material

such as plastic, rubber or wax. xamples are carbon in ndian and

printers inks and in rubber, and titanium dioxide in white paint. n

biology the word pigment is much more loosely used; it includes

insoluble materials that may be colored or visible by virtue o being

reractile or bireringent. igments in the biological sense include

chlorophyll and the anthocyanins and carotenoids responsible or

colors other than green in plants. hese plant pigments dissolve in

alcohol and water, and are not seen in parain sections. emoglobin,

the colored oxygen-carrying metalloprotein o blood, is insolubilized

by fxation. s a basic protein, hemoglobin stains strongly with anionic

dyes such as eosin.

n pathology, abnormal insoluble deposits, yellow, brown or black

without staining, and not distinctively stained with &, are requently

encountered. igments play an important part in the diagnosis o

diseases and conditions such as gout, kidney and gallstones, jaun-

dice, melanomas, albinism, hemorrhage and tuberculosis. n a section

o tissue, the term pigment reers to a material that has color and

can be seen without staining. t can be either normal or pathological.

igments are identifed either by their color, size and shape or by

chemical testing. For example, i a chemical test that gives a blue

product is applied to a yellow pigment, the result may be a green

color. igments can be placed in three categories: artiacts o fxa-tion, exogenous and endogenous (able 1). ome fxative-induced

and endogenous pigments are described in this chapter.

Table 1. Common Pigments.

Fixation Artifacts Exogenous Pigments Endogenous Pigments

Formalin pigment

(in and near blood)

Black

Mercury pigment

(everywhere in the tissue)

Black

Picric acid

(everywhere in the tissue)

Yellow

Osmium dioxide

(in most parts of

tissues; darkest in fat

cells, lipid droplets)

Gray to black

Carbon

(in lungs and associated

lymph nodes, especially

of city dwellers,

coal miners)

Black

Inks used for tattoos

(skin)

Various colors

Melanosis coli

(lipofuscin-like deposits

in colonic mucosa

of habitual users of

anthraquinone purgatives)

Brown

Melanins

(in normal skin,

eye, some neurons;

melanomas)

Brown to black

Hemosiderin

(in cells that have

phagocytosed blood;

liver in diseases of

iron metabolism)

Dark yellow to brown

Lipofuscin

(in older people, in

cardiac muscle cells,

neurons etc.)

Yellow to light brown

Sodium urate

(in lesions of gout)

Not a pigment but

included here or

convenience


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Pigments and Mineralsigments and Minerals

ixationArtifacts

ormalinpigment,also called acid hematin, is ormed in specimens

xed in ormaldehyde solutions at p below 5.5, especially ater

everal weeks in such a solution. lkaline ormaldehyde (p above

) may cause the same problem. his pigment, a product o

egradation o hemoglobin, diuses or a short distance rom its

te o ormation and settles out as an insoluble product: abundant

mall black dots wherever blood is present in a section. his artiact

oes not occur when the fxative is a ne utral, buered ormaldehyde

olution. Formalin pigment can be removed by treating the sections

ith a saturated solution o picric acid in ethanol or two hours.

ercurypigmentconsists o abundant uniormly distributed tiny dark

rystals, probably containing mercurous chloride. hese deposits

ccur in all tissues fxed in liquids containing mercuric chloride,

cluding B5", eidenhains U and Zenkers uid. he material

removed by brie treatment o sections with an iodine solution,

llowed by sodium thiosulate to remove the brown iodine stain.

EndogenousPigments

Melanins

elanins are brown to black (eumelanin) or yellowish (pheomelanin)

olymeric pigments ormed rom the amino acid tyrosine by a series

oxidations and other reactions (Figure 1) in skin cells, hair, eyes

etina, iris and choroid) and in the cell bodies o some neurons,

otably in the substantia nigra and locus coeruleus o the brain stem.

skin, melanocytes, branched cells at the junction o the epidermis

ith the dermis, synthesize the pigment and package it into protein-

ontaining granules called melanosomes. he melanosomes are

en extruded and taken up by epithelial cells in the deepest layer o

e epidermis, an event known as pigment donation. athologically,

elanin is ound in the cells o malignant melanomas and various

enign nevus tumors derived rom melanocytes.

elanins are not extracted by acid treatments that remove ormalin

gment. Melanins are, however, bleached by oxidation with an

queous solution o either hydrogen peroxide or potassium perman-

anate, allowed to act or 12 to 24 ho urs. he latter reagent must be

llowed by oxalic acid, to remove the brown manganese dioxide

that deposits on the sections. istochemical staining o melanin is

accomplished by exploiting the chemical reducing properties o this

pigment. wo methods are commonly used: he Masson-Fontana

silver method and chmorls erric erricyanide reaction.

n the Masson-Fontana technique, the slides are immersed in a

solution containing silver diammine, g(3)

2+, ions. his is made

by adding ammonium hydroxide (strong ammonia solution) dr opwise

to 5% aqueous silver nitrate. brown precipitate o silver oxide is

frst ormed, but dissolves as the diammine is ormed by addition o

more ammonia. Finally, a drop o silver nitrate is added, making the

solution opalescent (rom gO) and ensuring th at there is no excess

o 3. Reducing groups in tissues convert g(

3)

2+ to colloidal

metallic silver, which is brown or black. Optionally the color can be

intensifed by immersion in gold chloride, replacing the silver with

gold. he gold chloride o histology is either tetrachloroauric acid ,

ul4

or its sodium salt. contrasting counterstain such as neutral

red or the aluminum complex o nuclear ast red, is applied to show

the tissue architecture. he reactions o ormation and reduction o

silver diammine and gold toning are summarized in Figure 2.

ubstances in tissues that reduce silver diammine are said to be

argentaffin, they include melanin and phenolic compounds, notably

5-hydroxytryptamine in certain endocrine cells o the epithelium o

the stomach and small intestine, known as enterochromain cells.

n chmorls reaction, sections are immersed in a solution containing

erric chloride and potassium erricyanide. Melanin reduces erric

ions (Fe3+) to errous ions (Fe2+), and the errous iron then combines

with erricyanide. he expected product is errous erricyanide, a blue

pigment known as urnbulls blue. n act, the product is the same

as russian blue, which is ormed when erric ions combine with

errocyanide ions. russian blue is erric errocyanide, Fe4[Fe()

6]3,

occurring in crystals that also contain water molecules and sodium

or potassium ions. he color is associated with the occurrence o

iron in both oxidation states (+2 and +3) in the same molecule. he

fnal step is application o aluminum-nuclear ast red or a similar

counterstain. s with the Masson-Fontana method, this reaction is not

melanin-specifc and may stain other elements, such as argentain

and chromain cells and some types o lipouscin.

Figure 1. Biosynthesis o melanin, starting with the amino acid tyrosine. This simplifed scheme omits several intermediates and alternative metabolic pathways.

Several o the reactions occur without the need or catalysis by enzymes. This scheme applies only to eumelanin, the pigment in air, brown and black skin and hair.

Related pigments such as neuromelanin (in aminergic neurons) and pheomelanin (in red hair) are ormed by other metabolic transormations o dopa and dopachrome.


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Hemosiderin

emosiderin, an aggregate o proteins containing iron ions, is a yellow

to brown pigment seen in cells that have phagocytosed and degraded

hemoglobin. major component is erritin, a protein that orms hollow

particles about 10 nm in diameter, each containing 2000-4500 Fe3+

ions. Ferritin, which is not colored, is not the only component o

hemosiderin. Glycoproteins are also present, and the deposits give

a positive periodic acid-chi reaction. ome o the pathological

conditions with hemosiderin deposits are hemorrhages o any kind,

hemolytic anemia, some liver diseases, the lungs in congestive heart

ailure, and in the liver, pancreas and skin in hemochr omatosis. his

last condition is a group o metabolic diseases in which iron absor-

ption rom the small intestine, normally regulated by demand, is

uncontrolled. ron cannot be excreted, and the excess accumulates

as hemosiderin in macrophages.

emosiderin is insoluble in alkalis and soluble in either 0.4 M aqueous

oxalic acid (six hours) or 0.06M sodium dithionite in acetate buer,

p 4.5 (fve minutes). hese treatments remove the visible unstained

pigment and prevent histochemical staining, which is based on

detection o iron by ormation o russian blue, using the method

o erls.

he histochemical reaction or protein-bound iron traditionally had

two stages: release o erric ions by denaturing the binding proteins

with hydrochloric acid, ollowed by treatment with potassium erri-

cyanide solution to produce russian blue. iusion o the released

Fe3+ resulted in blue deposits around the pigment-containing cells. n

1867, Max erls devised a stable reagent containing con centrations

o acid and errocyanide that optimally precipitated the Fe3+ within the

cells. russian blue is insoluble in acids but soluble in alkalis. he

commonly used red counterstains are applied rom acid solutions;

neutral red and the aluminum complex o nuclear ast red both contrast

well with russian blue. osin can be used i contrast between nuclei,

cytoplasm and collagen is not needed. Figure 3 shows iron- containing

cells in a section o bone marrow.


Figure 3. Perls method demonstrates iron

(blue) in this bone marrow specimen. The

counterstain is aluminum-nuclear ast red

gure 2. Chemical reactions in the ormation o silver diammine (ammoniacal

ver nitrate), its reduction in sections o tissue, and gold toning.

Most o the iron in any vertebrate animal is in the heme o hemoglobin,

so tightly bound that it cannot be released or staining by any method

that would not destroy a section on a slide. istochemical staining

or iron with erls method is thereore tantamount to staining

hemosiderin. here are chemical tricks to enhance the sensitivity,

allowing staining o normal cells containing iron-binding and iron-

transporting proteins, such as the duodenal epithelial cells through

which iron is absorbed. he simplest o these is Quinckes method,

which dates rom 1887. lides are frst immersed in an ammonium

sulfde solution (malodorous, alkaline, removes sections rom slides),

which attacks the metal-binding proteins, reduces Fe3+ to Fe2+ and

immediately precipitates errous sulfde, Fe, at the sites o reduction.

Fe is an almost black compound, but it shows as gray in sections

5-10 m thick. ubsequent immersion in a potassium erricyanide

solution generates a blue pigment, simplistically urnbulls blue but

actually the chemically identical russian blue. t is also possible

to ampliy the blue deposits, taking advantage o the act that they

catalyze the oxidation o 3,3'-diaminobenzidine (B) by hydrogen

peroxide. he brown polymer ormed by oxidation o B is per-

manent, whereas russian blue ades ater several months.

Lipofuscin

ipouscin is a yellow-brown to reddish-brown pigment, ound within

cells in many parts o the body. he nickname wear-and-tear

pigment (Abnutzungspigment) reects the accumulation o lipouscin

with advancing age in cells that are either terminally dierentiated

(e.g. cardiac muscle fbers, neurons) or are inrequently replaced

(e.g. adrenal cortex, liver). he pigment is ormed rom ragments o

membranous organelles, which become permanently sequestered

in lysosomes. his accumulation o lipouscin does not appear to

interere with cellular unction. athologically, lipouscin is present in

the neuronal ceroid lipouscinoses, a group o several rare inherited

disorders within the large category o lysosomal storage diseases.

ormal lipouscin contains atty acids that are closely associated with

protein. his association allows most o the pigment t o remain in place

during passage through the solvents used in parain embedding

he staining properties o lipouscin are attributable to hydrophobic

character, the presence o unsaturated (=) linkages

and o glycols and aldehydes produced by their oxidation, and the

presence o weak acid (carboxy, phosphate) groups. ierent types o

lipouscin, including adrenal lipouscin, cardiac lipouscin, hemouscin

and ceroid, have been described on the basis o applying a panel o

staining methods that detect these physical and chemical properties.


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is recommended that more than one special staining technique be

erormed to confrm the presence o lipouscin and to distinguish

rom hemosiderin or other pigments that might also be present in

e tissue.

ome staining techniques applicable to parain sections are listed

able 2.

Urates

wo purines, adenine and guanine, constitute hal o the nucleotide

bases o and R. he purines released rom dead cells are

largely salvaged and incorporated into new nucleotides, but small

quantities are transormed to xanthine, which is oxidized to uric acid.

hese enzyme-catalyzed reactions occur principally in the liver; the

uric acid is released into the blood and excreted in the urine. n

increased circulating level o uric acid (hyperuricemia) can result

rom metabolic errors in the recycling o purines or rom insuicient

excretion by the kidneys. n saline solutions at physiological p, 99%

o uric acid is present as the biurate ion. odium biurate (monosodium

urate) is, however, sparingly soluble (5 to 10 mg/100 ml, d epending

on the method o measurement). his is not much higher than the

normal concentration in blood and extracellular uids (3 to 7 mg/

100 ml), and hyperuricemia is associated with the precipitation o

crystals o sodium biurate, which orm principally in joint and in the

kidneys, a condition known as gout. arger accumulations o the

crystals in sot tissues, especially near joints, are known as tophi.

espite their low solubility, sodium biurate crystals can be dissolved

by aqueous fxatives, so specimens are fxed in 95% ethanol. xam-

ination with polarizing optics shows needle-like bireringe nt crystals.

istochemical identifcation is possible because uric acid is a strong

reducing agent (Figure 5). t can thereore be demonstrated using

the two methods discussed or melanin; urate crystals reduce

silver diammine or erric erricyanide more rapidly than other tissue

components. Gomoris methenamine-silver method, can also be

used to identiy urate cr ystals in tissue sections. ts action is slower.

able 2. Some properties of lipofuscin.

eroid is a type o lipouscin with the additional property o acid-ast staining.

Staining MethodAppearance

of Lipofuscin

Explanation

None Brown, autofuorescent;

not bleached by H2O

2

Possibly rom

compounds ormed by

reaction o aldehydes

with amino groups, and

rom favoproteins

Oil red O Red Anity o hydrophobic

dye or lipid

Sudan black B Black Anity o hydrophobic

dye or lipid

Cationic dyes (azure A,

ethyl green etc, pH 4.

Positive Carboxy groups o atty

acids

PAS Pink-purple Aldehydes rom oxidized

sites o unsaturation

Ziehl-Nielsen stain (basic

uchsine with phenol,

ollowed by acid-alcohol)

Ordinary lipouscin

negative. Red acid-ast

staining o ceroid1

Lipoprotein retards

extraction o cationic dye

bound to atty acids

M as son-F ont an a Brow n; v ari abl e Al deh yd es ro m ox idi ze d

sites o unsaturation

Schmorls reaction Blue -green Uncert ain identit y o

reducing groups

Oxidation ollowed by

aldehyde-uchsine

P ur pl e ( Fi gu re 4) P ro ba bl y c ar bo xy gr ou ps

generated rom oxidized

unsaturated sites

Figure 4. Lipouscin in cells o the liver

Gomoris aldehyde-uschsine, applied ate

oxidation with potassium permanganate

and treatment with oxalic acid to remove

deposited MnO2. The counterstain is tar-

trazine, a yellow anionic dye.

Figure 5. Formation o uric acid and sodium

biurate rom purines, and the chemical

oxidation o uric acid. The enzyme urate

oxidase occurs in all mammals other than

monkeys, apes and man. Sodium urate

crystals in tissues are easily oxidized by

silver diammine or erric erricyanide, with

concomitant deposition o silver (black) or

Prussian blue.


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Pigments and MineralsPigments and Minerals

ilePigments

uman red blood cells have an average liespan o 120 days, ater

hich they are degraded in the spleen and bone marrow, and most

their components are recycled and used in the production o

ew red blood cells. Removal o iron rom the heme o hemoglobin

esults in the ormation o biliverdin, a green compound. Biliverdin is

ansported to the liver, where is it urther reduced to bilirubin, which

as an orange color. Bilirubin is then removed rom circulation in

e blood and secreted into the duodenum as a component o bile.

iliverdin and bilirubin are considered bile pigments. Bile pigments

an vary in color rom yellowish-brown to green. athologically, excess

le pigment is seen in patients with liver ailure, hemolytic anemia, or

hen there is an obstruction in the ow o bile rom the liver. ll these

onditions are associated with jaundice, a yellow coloration o t he skin

ue to bilirubin. n the liver, bile pigments appear in hepatocytes as

ellow-brown globules. For the pathologist, it is sometimes necessary

distinguish bile pigments rom lipouscin, particularly in cases o

ossible sepsis in patients with liver transplants. ematoidin is a

rown pigment similar but not identical to bilirubin, ound in sites o

emorrhage or inarction.

raditional tests or bile pigments in tissue were based on the test

developed by eopold Gmelin (1788-1853) or detecting bilirubin in

urine: transient colors (green, blue, violet, red, yellow) orm during

oxidation by concentrated nitric acid. nother traditional method is

ndr Fouchets reaction, frst introduced in 1917, also or urine.

dapted as a histochemical method by M.J.all in 1960, this provides

the most reliable and reproducible special staining technique or

demonstrating bile pigments. t is applied to parain sections o

ormaldehyde-fxed tissue. Bilirubin is oxidized to biliverdin by treating

the sections with Fouchets reagent, a solution containing erric

chloride and trichloracetic acid. he sections are then counterstained

with Van Giesons solution. Only bile and bile pigments in the liver

are detected, when stained green with this method. ematoidin in

other locations does not give the reaction. (Van Giesons solution is

0.1% acid uchsine in saturated aqueous picric acid; it colors collagen

fbers red and everything else in the section yellow.)

mall amounts o bile pigments are lost during routine tissue pro-

cessing and staining because o their slight solubility in organic

solvents. arge deposits o bile pigments, however, can resist these

processing procedures. t is recommended that two known positive

control sections be processed with the test section. Both sections

should be oxidized with Fouchets reagent, but only one should be

counterstained with Van Giesons mixture. Fouchets reagent should

be prepared on the day it is to be used.

Minerals

n histology and histopathology the word minerals is applied to

substances detected by orming colored reaction products specifc

to metal ions or inorganic anions. he most common minerals that can

be demonstrated by special staining techniques are calcium, iron and

copper. he previous section detailing identifcation o hemosiderin

encompasses the detection o iron. pecial staining techniques or

calcium and copper are now discussed.

Calcium

alcium is present in hydroxyapatite, a10

(O4)

6(O)

2, the insoluble

mineral o bones and teeth. bnormal deposits o calcium phosphate

or carbonate can be associated with necrotic tissue in lesions o

atherosclerosis, hyperparathyroidism, nephrocalcinosis, sarcoidosis,

tuberculosis, and in some tumors. alcium phosphate crystals can

orm in the cartilage o joints in a condition known as chondrocalcinosis

or pseudogout. cidic fxatives such as B ouins uid have the potential

to dissolve calcifed deposits and must thereore be avoided. eutral

buered ormaldehyde is suitable. here are many ways to stain

calcium, but only two methods are routinely used in histopathology.

hese are alizarin red and the von Kossa technique.

lizarin red is an anionic anthraquinone dye that orms sparingly

soluble salts with calcium ions (Figure 6). n the original techniques

published in the 1950s (methods o ahl and o McGee-Russell)

alizarin red is used at p 4.8 or 6.1 and gives an orange color with

calcifed deposits. he yellow component is attributed to impurities in

the dye. Moreover, diusion o the colored product is usually evident,

indicating partial dissolving o calcium phosphate or carbonate beore

precipitation o a2+ by the dye. mino groups o proteins in the tissue

also bind the dye and must be removed by dierentiation, leaving a

pink background stain.

pplication o alizarin red at p 9 (method o uchtler) all ows both

the sulphonate group on carbon 3 and the ionized hydroxy group on

carbon 2 to participate in salt ormation with calcium (Figure 6). he

impurities do not react at this higher p, and the resulting calcium

salt has a deep red color. n alkaline p also suppresses protonation

o amino groups, preventing most o the background staining and

obviating the need or dierentiation. disadvantage o staining with

any alkaline solution is the risk o sections detaching rom the slides

staining is carried out at p 4.8 or 6.1, the reaction must be moni-

tored microscopically and stopped, usually ater one or two minutes,

beore diusion artiacts appear. With the stain at p 9 diusion does

not occur and the slides may, with advantage, be let in the solution

or an hour.

Figure 6. Combinations o calcium ions

with alizarin red S. An earlier notion o

chelation o Ca2+ by oxygen atoms on

carbons 1 and 9 is no longer accepted.


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he von Kossa method indirectly localizes calcium in tissue by

etecting phosphate or carbonate ions. he sections are placed in

% aqueous silver nitrate. alcium cations are r eplaced by silver, with

ansormation o a3(O

4)

2to g

3O

4and o aO

3to g

2O

3. Both

lver salts are easily reduced to the metal, a reaction most easily

ccomplished by placing the staining dish u nder a 100W light bulb or

n a window sill. alcifed material is blackened in about 15 minutes

nd is sharply delineated. he sections are next immersed or a ew

inutes in a sodium thiosulate solution, which removes silver that has

omplexed with protein and would eventually darken with storage o

e slides. light red counterstain such as neutral red or aluminum-

uclear ast red contrasts well with the silver deposits.

he only objects likely to be conused with sites o calcifcation in a

on Kossa preparation are sodium biurate crystals, which reduce

lver nitrate even in the absence o light. Urate crystals can be

xtracted with alkali beore staining; a saturated aqueous solution

lithium carbonate (30 minutes) is used or this purpose because

hium biurate is more soluble in water than the corresponding sodium

r potassium salts.

ilver nitrate is an expensive compound, but the solution may be

eused and kept or several years in a brown glass bottle, provided

at it is not contaminated with organic matter (such as bits o

ssue sections) or with chloride, bicarbonate, carbonate, hydroxide,

hosphate or sulfde ions (rom inadequately cleaned glassware). ap

ater kills silver nitrate solutions.

nother calcium compound that can orm crystals in tissues is calcium

xalate. his condition, oxalosis, may be an inherited metabolic

sease (rare), a consequence o renal ailure or o poisoning by

xalate or ethylene glycol. alcium oxalate crystals also occur in

ssociation withAspergillus ungal inections in people with impaired

mmune unction. he crystals are bireringent and not dissolved by

cetic acid. he calcium is so tightly bound that it does not stain with

izarin red . he von Kossa reaction is positive, but the best staining

ethod is Yasues technique. ections are treated with 5% acetic

cid to remove calcium carbonate and phosphate, then transerred to

% aqueous silver nitrate. ilver displaces calcium ions and the

esulting silver oxalate is stained with dithiooxamide, a reagent that

combines with silver ions to orm a dark brown polymeric chelate.

ithiooxamide will be discussed later as a histochemical reagent

or copper.

Copper

opper is an essential nutrient, being a component o cytochromes

and many oxidoreductase enzymes. athologically, the accumulation

o copper is associated with Wilsons disease. his is a recessively

inherited metabolic disorder in which a transport er protein in liver cells

ails to move copper into the bile and ails to combine copper with

ceruloplasmin, the copper-binding protein o blood plasma. opper,

associated with albumin and other proteins, accumulates in cells

o the liver, cornea and corpus striatum o the brain in patients with

Wilsons disease. opper accumulations are seen also in primary

biliary cirrhosis and some other liver disorders.

wo reagents are suitable or histochemical demonstration o copper

by virtue o ormation o colored complexes. ithiooxamide (also known

as rubeanic acid) gives a stable dark green polymeric product that

can be mounted in a resinous medium.p-dimethylaminobenzylidene

rhodanine (MBR) gives a red product that dissolves in organic

solvents and thereore requires an aqueous mounting medium. he

structures o the compounds are shown in Figure 7. Both require

long (overnight) incubation to develop the colors. MBR is a more

sensitive reagent than dithiooxamide and is generally preerred

(Figure 8). he sensitivity o the dithiooxamide method can be

increased by prolonging the incubation to 72 hours.

Figure 7. Dithiooxamide (rubeanic acid)

and DMABR. The latter reagent is re-

quently and wrongly called rhodanine

but rhodanine is a dierent compound

that cannot be used or histochemical

staining o copper. Several xanthene dyes

with names that include rhodamine are

likewise unrelated.

Figure 8. A section o liver stained by

the DMABR method or copper (red) and

counterstained with hemalum (blue).


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Pigments and Minerals

FurtherReading

his list includes books, review articles and a small selection o original

papers.

Books and Reviews

Bentley KW. The Natural Pigments ew York: nterscience, 1960.

hurukian J. igments and Minerals. hapter 13 in Bancrot J, Gamble M

(eds). Theory and Practice of Histological Techniques, 5th ed. ondon:

hurchill ivingstone. pp. 243-267, 2002.

Kiernan J. Histological and Histochemical Methods: Theory and Practice

4th ed. Bloxham, UK: cion. (hapter 13, pp. 337-353), 2008.

illie R, Fullmer M. Histopathologic Technic and Practical Histochemistry

4th ed. ew York: McGraw-ill. (hapters 11 & 12, pp. 485-557), 1976.

earse G. Histochemistry, Theoretical and Applied 4th ed. dinburgh:hurchill ivingstone. (Vol. 2, hapter 20, pp. 973-1033), 1985.

rota G. he chemistry o melanins and melanogenesis. Progress in the

Chemistry of Organic Natural Products 1995;64:93-148.

Raper . omplexes o heterocyclic thione donors. Coord Chem Rev

1985;61:115-184.

lominski , obin J, hibahara , Wortsman J. Melanin pigmentation in

mammalian skin and its hormonal regulation. Physiol Rev2004;84:1155-1228.

Original Papers

Barrett M. On the removal o ormaldehyde-produced precipitate rom

sections.J Pathol Bacteriol1944;56(1):135-136.

hio K, Reiss U, Fletcher B, appel . eroxidation o subcellular organ-

elles: ormation o lipouscin-like pigments. Science 1969;166:1535-1536.

earing VJ, sukamoto K. nzymatic control o pigmentation in mammals.

FASEB Journal1991;5:2902-2909.

rons R, chenk , ee K. ytochemical methods or copper. Arch

Pathol Lab Med 1977;101:298-301.

illie R, izzolato . istochemical azo coupling reac tions o the pigments o

obstructive icterus and o hematoidin. . iazonium salts used. J Histochem

Cytochem 1969;17(11):738-748.

Mochizuki Y, ark MK, Mori , Kawashima . he dierence in autouorescenc

eatures o lipouscin between brain and adrenal. Zool Sci1995;12:283-288.

abuccuoglu U. spects o oxalosis associated with aspergillosis in pathology

specimens. Pathology Research and Practice 2005;201:363-368.

uchtler , Meloan , erry M. On the history and mechanism o alizarin

and alizarin red stains or calcium.J Histochem Cytochem 1969;17:110-124.

hornburg , Beissenherz M, olan M, Raisbeck MF. istochemical

demonstration o copper and copper-associated protein in the canine liver.

Vet Pathol1985;22:327-332.

Wilcox WR, Khala , Weinberger , Kippen , Klinenberg JR. olubility o

uric acid and monosodium urate. Mol & Biol Eng 1972;10:522-531.

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