Histochemistry

TechniquesAlcian Blue/PASAlkaline Congo RedBielschowskyBleaching Of MelaninBouinCAB(Chromotrope AnilineBlue)DescalcificadorEDTA neutralEosin-Phloxine Fite-Faraco(Wade Fite)FouchetGiemsa(Biopsy)Giemsa(Cytology)

Giemsa(H. Pylori)GMS (Grocott)GMS(basal membrane)GomoriGramGrimeliusMasson FontanaMasson ThrichromeMayer's HaematoxylinModified GiemsaMucicarmimOrceinPapanicolaou (cytology)PASPerl'sPTAHRehydration of air-dried

smearsResorcin-FucsinSafranin O for CartilageSafranin(Thrichrome)Sudan Black(or Oil Red O)Toluidine Blue(for MastCells)UNNA(Methyl Green Pyronin)Van GiesonVon KossaWarthin-StarryWeil's(Myelin)Zhiel-Neelsen

Alcian Blue/PAS

1 ) Dewax sections and bring towater

2) Alcian Blue 5m 3) H2O d 4) Periodic Acid 1% 5m 5) H2O d 6) Schiff's reagent 15m 7) Wash in water 8) Rinse in absolute alcohol 9) Xylene and Mount Results:

Acid-Mucins-Blue Neutral

Mucins-Magenta Mixtures of theabove-the colour will depend on thedominant entity and will range fromblue-purple through purple to aviolet or mauve colour

Solutions A)Alcian blue 1g 3% acetic

acid 100 cm B)Schiff's reagent Dissolve 1g

basic fuchsin in 200 cm of boilingdistilled water removing the flaskof water from the bunsen justbefore adding the basic fuchsinAllow the solution to cool to 50 Cand add 2g potassium metabisulphitewith mixing Allow to cool to roomtemperature then add 2cmconcentrated hydrochloric acid

mix add 2g activated charcoal andleave overnight in the dark at roomtemperature Filter through a N1Whatman paper when the solutionshould be either clear or a paleyellow colour Store in a darkcontainer at 4 C

Alkaline Congo Red

1)Sections to distilled water 2)Haematoxylin 3 min 3) Tap water until blue 4) Solution 1 (50ml sodium

chloride Saturated solution + 0.5mlsodium hidroxid 1%) 20m

5) Solution 2 (50ml Congo

Red/Sodium chloride Saturatedsolution + 0.5ml sodium hidroxide1%) 20 min

6) Wash in alcohol 80%-95%-

100%-Xylene 7) Mount

Results: Amyloid elastictissue eosinophil granules-rednuclei-blue Must use control

Bielschowsky

1 ) Dewax and bring sections towater

2) Wash well in distilled water

1-2m 3) Place sections in 2% silver

nitrate in the dark at 37 C for25m


2-3m 5) Reduce in 10% formalin

solution until turn yellow 6) Wash well in distilled water

3-5m 7) Place sections in ammoniacal

silver solution 20-40m 8) Treat with 10% aqueous

formalin solution until ten brown-yellow


3-5m 10) Tone in 0.2% gold chloride

solution 3-5m 11) Wash well in distilled

water 1-2m 12) Treat with 5% sodium

thiosulphate solution 3-5m 13) Wash in tap water 3-5m 14) Dehydrate clear and mount Result:

Axous-black Ammoniacal Silver Solutions: 20% silver nitrate solution

15ml mixed with 10ml AbsoluteAlcohol Add strong ammonia to mixedsolution until the initialprecipitate disappears After thatadd 5 drops of ammonia and filterbefore use

Bleaching Of Melanin

1) Potassium Permaganate 1%-3 m2) Wash in tap water

3) Oxalic acid 2%-until the

section is clear

Bouin

ac. Pcrico 75ml formol 25ml ac. Actico 5ml

CAB(ChromotropeAniline Blue)


2) Stain in Weigert's

haematoxylin 5m 3) Wash in tap water 5m 4) Stain in 1% Fosfomolibdic

Acid 2m 5) Wash in tap water 6) Stain in Chromotrope Aniline

Blue solution for 8m 7) Wash in tap water

8) Dehydrate clear and Mount Results Nuclei-blue Collagen-blue

Hepatitis-red Solutions: Chromotrope 2R 4g Aniline Blue 1g 0.2N HCL 200ml Put Aniline Blue in HCL and

warm a little After cool addchromotrope The PH should be 0.9-1.3

Descalcificador

Nitric Acid 10ml Formalin 10ml H2Od 80ml

EDTA neutral

EDTA disodium salt 250g Distilled water 1750cm This solution is often cloudy

It is neutralised to PH 7 by theaddition of approximately 25g ofsodium hydroxide which will alsoclear it.

Eosin-Phloxine

1) Eosin (stock solution) 100ml

2) Phloxine (stock solution) 10

ml 3) Alcohol 95 780 ml 4) Glacial Acetic Acid 4ml Stock solutions Eosin y 1g H2Od 100 ml Phloxine B 1g H2Od 100 ml

Fite-Faraco(Wade Fite)

1) Dewax in turpentin 20 m 2) Blot until almost dry 3) Wash in tap water blot dry

5m 4) Stain in fuchsin solution

(from Zhiel 1-filter first) 30m 5) Wash in tap water blot dry

5m 6) Decolourise in 10% sulphuric

acid until white 5m 7) Wash in tap water blot dry

5m 8) Stain in haematoxylin (or

methylene blue) 9) Wash in tap water blot dry

10m 10) Clean in xylene and mount Results: M Leprae (and other

mycobacteria)-Red Nuclei-blue Solutions: Kinyoun Solution: Basic fuchisin 40g Phenol 80ml Ethanol (95%)

200ml Distilled water 1000ml Methylene blue 2g

Distilled water 100ml

Fouchet

1) Sections to distilled water 2) Stain in Fouchet's reagent

5m 3) Rinse in distilled water 4 ) Counterstain in Van Gieson

solution 5) Dehydrate through alcohol to

xylene and Mount Results: Bile pigments-blue-green

Muscle-yellow Collagen-red

Solutions: Fouchet reagent: 25%trichloracetic acid (aqueous) 25ml10% ferric chloride (aqueous) 25mlV a n Gieson: Saturated aqueouspicric acid solution 90ml

1% aqueous acid fuchsin

sol.10ml

Giemsa(Biopsy)

1 ) Dewax and bring sections towater 2) Put slides in May-grunwaldworking solution 20 minutes at 60C3) Put slides in Giemsa workingsoluton 40m at 60C

4) Differentiate in acetic acid

(0.5%) 5sec 5) Dehydrate in Alcohol/Acetone

(1:1) 6) Xylene and mount Results: Red blood cells-yellow

to pink Nuclei-dark blue Eosinophil granules-red Solutions:

Working solution (prepare fresh

each time) Stock May-Grunwald stain 10ml

H2Od 50ml Stock Giemsa stain 1.75ml H2Od 50ml

Giemsa(Cytology)

1) Air dry slides (do not fix) 2) Fix in alcohol 95 1m 3) Tap water 4) May-Grunwald Nile (pure) 10m 5) Tap water 6 ) Giemsa Diluted 1/6 in

distilled water 15m 7) Tap water 8) Air dry and mount Results: Red blood cells-yellow to pink

Nuclei-dark blue Eosinophil Granules-red

Giemsa(H. Pylori)


2) Put slides in Giemsa 20%

30m 3) Wash in tap water 4) Differentiate in alcohol 95 5) Dehydrate and mount

GMS (Grocott)

1)Dewax and bring section towater

2)Oxidise in 5% chromic acid 1

hour (after 30m prepare solution(C) and put in oven at 60C)

3) Wash in tap water 4) Rinse in 1% sodium

bisulphite 5) Wash in tap water for 3m 6) Rinse well in several

changes of distilled water 7) Place in incubating solution

at 60C in the dark for 1hour

8) Wash well in many changes ofdistilled water

9) Tone in 0.1% gold chloride

for 4m 10) Place section in 3%sodium thiosulphate for 5m s

11) Wash well in tap water 12) Lightly counterstain in

light green solution 20 seconds 13) Wash in tap water 14) Dehydrate clear and mount RESULTS: Fungal hyphae and yeast bodies

- black Background - pale green

Solutions: a) 5% solution tebraborate in

distilled water (freshly prepared) b) 5% silver nitrate 5ml 3% methenamine (hexamine) 100ml Add the methenamine solution to

the silver nitrate solution awhite precipitate forms but clearson shaking (solution "a" and "b"keep well at 4C)

c) Incubating solution Borax solution "a" 5ml Distilled water 25ml Methenamine silver "b" 25ml

d) Light green counterstain Light green 100mg Acetic acid 0.1ml Distilled water 200ml

GMS(basal membrane)

1 ) Dewax and bring section towater

2 ) Oxidise in 1% periodic acid

for 15m 3) Wash in tap water for 3m 4) Rinse in 1% sodium

bisulphite 5) Wash in tap water for 3m 6) Rinse well in several

changes of distilled water 7) Place in incubating solution

at 60 C in the dark for 1hour 8) Wash well in many changes of

distilled water 9) Tone in 0.1% gold chloride

for 4m 10) Place section in 3% sodium

thiosulphate for 5m 11) Wash well in tap water 12) Lightly counterstain in

light green solution 20secod 13) Wash in tap water 14) Dehydrate clear and mount Results: Basal Membrane-black

Background-pale green Solutions:

a) 5% solution tebraborate in

distilled water (freshly prepared) b) 5% silver nitrate 5ml 3% methenamine (hexamine) 100ml

Add the methenamine solution to thesilver nitrate solution a whiteprecipitate forms but clears onshaking (solution "a" and "b" keepwell at 4C)

c) Incubating solution Borax solution "a" 5ml Distilled water 25ml Methenamine silver "b" 25ml d) Light green counterstain

Light green 100mg Acetic acid 0.1ml Distilled water 200ml

Gomori

1 ) Dewax section and bring towater

2) Treat with 1% potassium

permangante solution 2m 3) Rise in tap water 4) Bleach in 2% potassium

metabisulphite solution 5) Rinse in tap water 6) Treat with 2% iron alum 5m 7) Wash in several changes of

distilled water 8) Place in silver solution 1m

9) Wash in several changes ofdistilled water

10) Reduce in 4% aqueous

formalin solution 4m 11) Wash in distilled water 12) Tone in 0.2% gold chloride

solution 10m 13) Wash in distilled water 14) Treat with 2% potassium

metabisulphite solution 1m 15) Wash in distilled water 16) Treat with 2% sodium

thiosulphate solution 1m 17) Wash in distilled water

1 8 ) Counterstain with vangieson or light green

19) Dehydrate clear and mount Results: Reticular fibres-black Nuclei-grey Other tissue-according to

counterstain Solutions: 1) 1% potassium permanganate in

distilled water 2) 2% potassium metabisulphite

in distilled water 3) 2% iron alum in Distilled

water 4) 4% formalin(in H2Od) 5) 0.2% gold chloride in

distilled water 6) 2% sodiumthiosulphate in distilled water

7) Silver solution To 10ml of 10% potassium

hydroxide solution add 40ml of 10%silver nitrate solution allow theprecipitate to settle and decantthe supernatant Wash theprecipitate several times withdistilled water Add ammonia drop bydrop until precipitate has justdissolved Add further 10% silvernitrate solution until a littleprecipitate remains. Dilute to 100ml and filter Store in a darkbottle

Gram


2) Cover the slide with sol 11m

pour off 3) Rinse with sol 2 4) Cover the slide with sol 2

1m 5) Rinse with D W about 5sec 6) Cover the slide with sol 3

20-60sec 7) Rinse the D W about 5sec 8) Cover the slide with sol 5

1m

9) Rinse the D W about 5sec 10) Leave to air dry Mounting Results: Gram-positive microorganisms-

blue Gram-negative microorganisms-pink to red

Grimelius

1) Rehydrate sections throughgraded alcohols to distilled water

2) Place sections in preheated

silver solution at 60C for 3hours

3) Remove sections and drain

thoroughly 4) Place sections in freshly

prepared reducing solution at 45Cfor 1 minute

5) Rinse sections in distilled

water 6) Examine the sections

microscopically to checkimpregnation If the sections are

under-impregnated return to thesilver bath for a further 5-10 m

7) Drain sections and repeat

reduction 8) Rinse in distilled water for

1m 9) Wash well in tap water 10) Dehydrate clear and mount

in DPX Results: Argyrophil granules-

brown/black Neither insulin norsomatostatin containing granuleswill stain with this method

Solutions:

A)Silver solution 1% aqueoussilver nitrate freshly made 3 cm

Acetate buffer (PH 5.6) 10 cm

Double-distilled water 87 cmB)Reducing solution

Hydroquinone 1g Sodium sulphite crystals 5g Distilled water 100 cm

Masson Fontana

1) Sections to water 2) Treat with silver solution

for 45 m at 37C 3) Wash well in several changes

of distilled water 4) Treat with 5% sodium

thiosulphate for 2m 5) Wash in tap water 6 ) Counterstain in light green

for 2m 7) Wash in tap water 8) Dehydrate throug graded

alcohol to xylene 9) Mounting

Results: Melanin-black Argentaffin-black Chromaffin-black Some lipofucsins-black Nuclei-green Solutions: Silver solution: To 20cm of 10% silver nitrate

add concentrated ammonia drop bydrop until the formed precipitatealmost dissolves A faintopalescence is evident when the endpoint is reached If too much

ammonia is added 10% silver nitratecan be added drop by drop until thefaint opalescence obtained To thissilver solution add 20cm ofdistilled water and filter Stone ina dark bottle The solution willlast for about four weeks

Masson Thrichrome


2) Stain in Weigert's

haematoxylin 5m and differentiatein HCl 1% in alcohol 70%

3) Wash well in tap water 4) Stain in acid fuchsin

solution and ponceau de xilidinesolution 1:2 for 5m

5) Treat with phosphomolybdic

acid solution 1min 6) Stain in methyl blue or

light green solution 2m 7) Treat with 1% acetic acid

solution 1m 8) Wash in H2Od Alcohol 100

clear and mount Results: Nuclei-blue to black Cytoplasm muscle and

erythrocytes-red Collagen-blue or green Solutions: 1)Weigert's haematoxylin:

A)Haematoxylin 1g Absolute alcohol 100ml B)30% ferric chloride 4ml

Hydrochloric acid 1ml

Distilled water 95ml Solution "a" and "b" mixed

immediately before use with 1:12)Acid fuchsin solution:

Acid fuchsin 0.5g Glacial acetic acid 0.5ml Distilled water 100ml 3 ) Ponceau de xilidine

solution: Ponceau de xilidine(ponceau 2R)1g Glacial aceticacid 1ml

Distilled water 100 ml 4) 1% phosphomolybdic acid in

distilled water

5) Methyl blue solution: Methyl blue 2g Glacial acetic acid 2g Distilled water 100ml 6) Light green solution: Light green 1g Glacial acetic acid 1ml Distilled water 100ml 7) 1% acetic acid in distilled

water

Mayer's Haematoxylin

Haematoxylin 1g Distilled water 1000cm Potassium or ammonium alum 50g

(12H2O 91.8g) Citric acid 1.09g Chloral hydrate 50 g Sodium iodate 0.2g 1- Dissolve the alum in the

distilled water 2- When complete add the

haematoxilin 3- When all the haematoxilin

has dissolved add the sodium iodateand let stir for 10m

4- Add the citric acid and stir

for more 10m 5- Add the chloral hydrate and

stir till dissolved No need to filter.Ready to use. To test just put a few drops

into warm water they should turnblue immediately.

Modified Giemsa


2) Stain in 2% Giemsa for 30 m 3) Wash in tap water quickly 4) Air dry and bring to xylene 5) Mount Result: Helicobacter pyloric-

blue Solutions: Giemsa's stain 2g Distilled water 100ml

Mucicarmim

1)Take sections to water 2)Hematoxylin 5m 3) Tap water 4) Diferenciate in alcohol-Acid

2m 5 ) Mucicarmim solution

(filtrate before use) 30m 6) Distilled water quickly 7) Metanyl yellow 1m 8) Distilled water 9) Dehydrate clear and mount

Results: Mucin-dark pink to red

Criptococus capsul-dark pink to redNucleo-black Other tissues-yellowSolutions:

Carmin CI n 75450 1g Aluminium chloride anidro 0.5g

Distilled water 2ml Alcohol 50% 100ml Mix all together and boile for

more or less 2m until turn drakAfter cold filtrate and put inrefrigerator Yellow metanil:

Metanil yellow 0.25g Distilled water 100ml

Acetic acid glacial 0.25ml

Orcein

1)Dewax and bring section towater

2)Treat with acid permanganate

solution 10m 3) Wash in tap water 4) Bleach with 2% aqueous

oxalic acid solution until clear 5) Wash in tap water 6) Rinse in 70% alcohol 7) Stain in Orcein solution at

room temperature 4hours 8) Rinse in 70% alcohol

9) Dehydrate clear and mount Results: Hepatitis B affected liver

cells-medium brown to blackBackground-clear to pale brown

Solutions: A) Acid permanganate solution

Potassium permanganate 1.5g Concentrated sulphuric acid 1.5ml Distilled water 100 ml

B) Orcein solution Orcein 2g 70% alcohol 100ml Concentrated hydrochloric acid

2ml

Papanicolaou (cytology)

1) Rinse slides in 95 %alcohol

2) Rinse slides in 70 %

alcohol 3) Rinse slides in distilled

water 4) Stain slides in Mayer's

haematoxylin for 3m 5) Wash in tap water to blue

haematoxylin 6) Rinse slides in 95% alcohol 7) Stain slides in OE6 for 3m 8) Rinse slides in 95% alcohol

twice 9) Stain slides in EA50 for 3m 10) Rinse slides in 95 %

alcohol twice 11) Rinse slides in absolute

alcohol twice 12) Rinse slides in xylene

twice 13) Mount in D P X Results: Nuclei-blue

Superficial cell cytoplasm-pinkIntermediate and parabasal cellcytoplasm-blue-green

PAS

1) Section to water 2) 0.5% aqueous periodic acid 5

m 3) Rinse in tap water and

distilled water 4) Stain with Schift's reagent

15m 5) Wash in running tap water

10m 6) Stain in haematoxylin 1min 7) Wash in running tap water 8) Dehydrate through alcohols

to xylene and mount

Results: A c i d mucin Neutral mucin-

magenta Nuclei-blue Solutions: 1) 0.5% periodic acid in

distilled water 2) Schiff's reagent: Basic fuchsin 1g Distilled water 200ml Potassium metabisulphite 2g

Hydrochloric acid 1N 2ml (1N=HCl8.35ml+H2Od 91.65ml)

Activated charcoal 2g

Dissolve 1g basic fuchsin in200ml of boiling distilled water removing the flask of water fromthe bunsen just before adding thebasic fuchsin Allow the solution tocool to 50C add 2g potassiummetabisulphite with mixing Allow tocool to room temperature then add2ml concentrated hydrochloric acid mix add 2g activated charcoal andleave overnight in the dark at roomtemperature Filter through a N 1Whatman paper when the solutionshould be either clear or a paleyellow colour Store in a darkcontainer at 4C

Perl's


2) Wash in distilled water 3) Transfer section to freshly

prepared incubating solution 10m 4) Wash in tap water 5) Counterstain in nuclear fast

red 6) Wash rapidly in tap water 7) Dehydrate through graded

alcohols to xylene 8) Mount

Results: Ferric iron-blue Nuclei-red

Solutions: A) Incubating solution: 2% hydrochloric acid 25ml 2% potassium ferrocyanide

25ml (Prepare fresh before use) B) Nuclear fast red Aluminium sulphate 5g Distilled water 100ml Nuclear fast red 100mg (boil for 5m)

PTAH

(Phosphotungstic Haematoxylin) 1 ) Dewax sections and bring to

water 2) Iron alumen 4% 20m-1hour 3) PTAH solution at 60 1-

2hours 4) Rinse in alcohol 95 (fast) 5) Alcohol 100 xylene and

mount Results: Muscle striations neuroglia

fibres fibrin amoebae-dark blue

Nuclei cilia red blood cells-blue Myelin-light blue

Collagen osteiod cartilage

elastic fibers-deep brownish redCytoplasm-pale pinkish brownParietal cell-pale blue

Solutions: P T A H Haematoxylin 0.5g

Phosphotungstic acid 10g H2Od 500ml Potassium permaganate(0.25%) 25ml Dissolve thehaematoxilin in 100 ml distilledwater and the acid in the remainingwater Mix the two together and addthe potassium permaganate Use after24 hours

Rehydration of air-driedsmears

1) Put slides in 50% aqueoussolution of glycerine 3m

2) Wash in 95% alcohol 2 times 3) Wash in tap water then in

H2Od 4) Stain in haematoxylin for

3m 5) Wash in tap water 6) Stain with eosina or Shorr 7) Wash in 9)5)% alcohol 8) Dehydrate clear and mount

Resorcin-Fucsin


2) Place in working solution

overnight (at least 12 hours) 3) Rinse in 80% alcohol 4) Wash in tap water 5 ) Counterstain in Van Gieson

3-5 m 6) Dehydrate clear and mount Results: Elastin fibre-deep purple

Collagen fibre-red Epithelial cellsand muscles-yellow

Stock Solution: 1)Dissolve 2g crystal violet

and 4g resorcinol in 100mldeionised water

2)Boil for 3m stirring

constantly with a combined magneticstirrer and hotplate

3) Add 15ml freshly prepared

30% aqueous anhydrous ferricchloride and bring back to theboil

4) Add 2g basic fuchsin or

pararosaniline (Product P1528;Sigma Poole Dorset UnitedKingdom) in 50 ml distilled watertogether with a further 15ml ferricchloride solution and boil again

5) Cool and filter 6) Retain the filtrate

(solution 1) and the filter paperwith its attached deposit

7) Place this filter paper and

its deposit in 150ml 90% alcoholand boil on a hotplate for 3m :then filter once more and retainboth the second filtrate and itsaccompanying filter paper withdeposit

8) Make the second filtrate up

to 150 ml with 95% alcohol and4.5ml concentrated hydrochloricacid and combine with solution 1

9) Add the second filter paper

together with its deposit 10) Bring to the boil on an

electric hotplate add a further 4gresorcinol and boil for three m :filter while hot The filtrateconstitutes the stock solution

Working solution Take 10 ml of

the stock solution and add 40 ml of50% alcohol Add 1ml concentratedhydrochloric acid

Safranin O for Cartilage

1) Sections to water 2) Stain with Mayer's

haematoxilina 3) Wash in runningtap water

4) Stain in SafraninO 5m 5) Dehydrate starting on

alcohol 95 and stay 2m on eachsolution to differentiate

6) Xylene and mount Results: Nuclei - blue Cartilage mucin

mast cell granules - orange to redSolutions

Safranin O (C I 50240) 0.1g H2Od 100ml

Safranin(Thrichrome)


2) Stain in haematoxylin 3m 3) Wash in running tap water 4) Stain in eosin 1m 5) Wash in 95% alcohol to 100%

alcohol 6) Stain in SafraninO solution

5m 7) Wash in 100% alcohol 8) Clear and mount Results: Cytoplasm-pink to

yellowish Solutions Safranin O 2g Absolute alcohol 100 ml

S udan Black(or Oil RedO)

1) Put frozen sections inalcohol 70% 1-2m

2) Stain in black sudan

saturated or oil red O (filtratesolution first and cover in a petridish) 15m

3) Wash in 70% alcohol unitl

almost no colour 4) Stain in haematoxylin 3m 5) Wash well in tap water 6) Rinse in distilled water 7) Mount in glicerine and close

around with nail polish Results: Stains unsaturated cholesterol

esters and trigrycerides-blue-blackSome phospholipids appear grey RedOil O: hydrophobic lipids andmineral oils are stained red Somephospholipids appear pink

Toluidine Blue(for MastCells)


2) Cover in Toluidine Blue 0.5%

Solution 30m 3) Wash in tap water quickly 4 ) Differenciate in 0.5%

Glacial acetic acid util only thecell nuclei and Mast cell arestained purple control under themicroscopy

5) Wash in tap water 6) Air dry Mounting

Results: Mast cell granules-purple Cell

nuclei-blue Solution: 0.5% Toluidine blue in H2Od

UNNA(Methyl GreenPyronin)

1)Dewax and bring sections towater

2)Rinse in acetate buffer PH

4.8 3) Place in staining solution

25m 4) Wash in tap water 5) Rinse fast in acetone 6) Acetone-xylene 1:1 xylene

and mount Results:

DNA-green-blue RNA-red Somemucous cells may be stained by thepyronin

Solution: Staining solution (Prepare

before use) Stock 2% methyl green 15cm

(Chloroform washed) Stock 2% pyronin Y 4cm Acetate buffer PH 4.8 23cm

Glycerol 14cm Acetate buffer A Stock solution

0.2M acetic acid (MW 60.05) 1.2cmglacial acetic acid in 100 cm H2Od

B Stock solution 0.2M sodium

acetate 1.64g sodium acetate

anhydrous (MW 82) or 2.72g sodiumacetate trihydrate (MW 136) in 100cm of distilled water

Buffer solution PH 4.8 20ml sol A 30ml sol B 50ml H2Od

Van Gieson


2) Stain in haematoxylin 3m 3) Wash in running tap water 4) Stain in Van Gieson solution

3m 5) Blot with paper 6) Dehydrate clear and mount Results: Nuclei-blue Collagen-red Other

tissue-yellow Solution Van Gieson solution:

Saturated aqueous picric acid

solution 50ml 1% aqueous acid fuchsin

solution 9ml Distilled water 50ml

Von Kossa

1) Section to distilled water 2) Place in silver nitrate

solution and expose to strong light60m

3) Wash in 3 changes of

distilled water 4) Treat with sodium

thiosulphate 5m 5) Wash well in distilled water 6 ) Counterstain in Van Gieson

2m 7) Dehydrate clear and mount Results:

Mineralised bone -black

Calcium-golden yellow Solutions: 1) 1% silver nitrate: Silver nitrate 1g Distilled water 100ml 2) 2.5% sodium thiosulphate: Sodium thiosulphate 2.5g Distilled water 100ml 3) Van Gieson: Saturated aqueous picric acid

solution 50ml

1% aqueous acid fuchsinsolution 9 ml Distilled water 50ml

Warthin-Starry


2) Buffer solution (a) 3) Solution b at 60 1hour 4) Solution c at 60 3m 5) H2OC at 60 6) Buffer solution 7) Dehydrate clear and mount Results: Spirochactes-black or dark

yellow-brown

Background-pale yellow to brown Solutions: A Buffer solution Sodium acetate 1.64g Acetic Acid 2.5ml H2Od 200ml B Silver solution Silver nitrate 0.5g Buffer solution (a) 50ml C Reduction solution a)Hydroquinon 300mg Buffer solution 10ml

Mix 3cc with 45cc of gelatin

(5%) and store at 37% b) Silver Nitrate 2% at 60

9ml Mix solution a) and b) before

use

Weil's(Myelin)


2) Stain in working solution 20

m 3) Tap water 4 ) Differenciate in 4% iron

alum 8 seconds (until thebackground is grey)

5) H2Od 6) Complete differentiation in

Weigert's Borax ferricyanidesolution 1sec

7) H2Od and tap water

8) Eosin 10sec 9) Dehydrate clear and mount Results: Myelin-dark grey to black

Background-pink Solutions: Working solution 4% iron alum and 1%

haematoxilin 1:1 Mix just beforeuse !!!

Weigert's Borax Ferricyanide

Solution Borax (Di-Sodium Tetraborate)

2g Potassium Ferricyanide 2.5g(if have 3H2O 2.86g)

H2Od 200ml

Zhiel-Neelsen

1 ) Dewax section and being towater

2) Wash in D W 3) Stain in Sol 1(dye red) 10m

(filter solution before use) 4) Wash in tap water well 5) Stain in Sol 2(dye blue) 5m

(filter solution before use) 6) Wash in tap water dry 7) Mounting Results: Bacilli-red Other tissues-blue

Alcian Blue/PASAlkaline Congo RedBielschowskyBleaching Of MelaninBouinCAB)DescalcificadorEDTA neutralEosin-PhloxineFite-Faraco)FouchetGiemsa)Giemsa)Giemsa)GMS )GMS)GomoriGramGrimeliusMasson FontanaMasson ThrichromeMayer's HaematoxylinModified GiemsaMucicarmimOrceinPapanicolaou )PASPerl'sPTAHRehydration of air-dried smearsResorcin-FucsinSafranin O for CartilageSafranin)Sudan Black)Toluidine Blue)UNNA)Van GiesonVon KossaWarthin-StarryWeil's)Zhiel-Neelsen

Histochemistry

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