-Dr Sowmya Srinivas

SPECIAL STAINS

CONNECTIVE TISSUE STAINSCARBOHYDRATE STAINSLIPID STAINSNUCLEIC ACIDS STAINSPROTEINSMICRO ORGANISMS

TYPES OF SPECIAL STAINS

Connective tissue (CT) is one of the four types of biological tissue that support, connect, or separate different types of tissues and organs in the body.

It develops from the mesoderm. The other three types are epithelial, muscle, and

nervous tissue. Connective tissue is found in between other tissues

everywhere in the body, including the nervous system.

All connective tissue apart from blood and lymph consists of three main components: fibers (elastic and collagenous fibers), ground substance and cells

CONNECTIVE TISSUE

The connective tissues are divided into the following groups:

Connective tissue proper – includes loose or areolar, dense, adipose, reticular

Cartilage – hyaline elastic and fibrocartilageBone – spongy or cancellous and dense or

corticalBlood Blood-forming – hematopoietic.

Connective tissue includes-CollagenElastinReticulinBasement membraneMuscle

CONNECTIVE TISSUE STAINS Collagen:Purpose: Bind bones and other tissues to

each other.Components: Alpha polypeptide chains.Location: tendon, ligament, skin, cornea,

cartilage, bone, blood vessels, gut, and intervertebral disc.

Collagen may be differentially stained by any of the following:

Van Gieson's stainMasson's trichrome stainMallory's trichrome stainAniline blue stainEosinReticulin stain

COLLAGEN STAINS

Van Gieson's Stain is a mixture of picric acid and acid fuchsin.

It is the simplest method of differential staining of collagen and other connective tissue.

It was introduced to histology by American neuropsychiatrist and pathologist Ira Van Gieson.

VAN GIESON’S STAIN

Solution:Saturated aqueous solution of picric acid

(approximately 1 %)- 100 ml1 % acid fuchsin - 10 ml The above quantities may be prepared and

kept as a stock solution, but it is better to use a freshly prepared solution containing 5 ml of saturated aqueous solution of picric acid and 0.75 ml of 1 % acid fuchsin which gives more precise and sharp staining.

STAINING TECHNIQUE1) Deparaffinise sections and bring to water.2) Stain with Weigert’s hematoxylin for 40

minutes.3) Wash in tap water.4) Differentiate in Acid alcohol.5) Wash well in tap water.6) Stain in Van Gieson stain for 3 minutes7) Blot and dehydrate through alcohols.8) Clear in Xylene and mount in permanent

mounting medium.

RESULTS:Nuclei – Blue/blackCollagen – RedOther tissues – Yellow

NOTE:Washing in water after Van Gieson solution

should be avoided, the colour balance being impaired by this.

Nuclear staining should be intense before application of Van Gieson solution; picric acid acts as a differentiator.

MASSON TRICHROME STAIN Fixation: Formal saline. Sections: All types.

Solution A:Acid Fuchsin – 0.5gGlacial acetic acid – 0.5mlDistilled water – 100ml

Solution B:Phosphomolybdic acid – 1gDistilled water – 100ml

Solution C:Methyl blue – 2gGlacial acetic acid – 2.5mlDistilled water – 100ml.

1) Deparaffinise sections and take to water.2) Remove mercury pigment by iodine, sodium

thiosulphate sequence.3) Wash in tap water.4) Stain nuclei by the celestine blue-

hematoxylin method.5) Differentiate with 1% acid alcohol.6) Wash well in tap water.7) Stain in acid fuchsin Solution A for 5 minutes.8) Rinse in distilled water.

METHOD

9) Treat with phosphomolybdic acid Solution B for 5 minutes.

10) Drain.11) Stain with methyl blue Solution C for 2-5

minutes.12) Rinse in distilled water.13) Treat with 1% acetic acid for 2 minutes.14) Dehydrate through ascending grades of

alcohol.15) Clear in Xylene, mount in permanent

mounting medium.

Nuclei – Blue/blackCytoplasm, muscle and RBC’s – RedCollagen – Blue.

RESULTS

Staining of Elastic tissue fibresVerhoeff's method Orcein technique Weigert’s Resorcin-fuchsin Aldehyde fuchsin method.

Verhoeff’s method is the classical method for elastic fibers and works well after all routine fixatives. Coarse fibers are intensely stained, but the staining of the fine fibers may be less than satisfactory. The differentiation step is critical to the success of this method

Preparation of stain: Solution aHematoxylin 5 gAbsolute alcohol 100 ml Solution bFerric chloride 10 gDistilled water 100 ml

Verhoeff’s method for elastic fibers

Solution c, Lugol’s iodine solutionIodine 1 gPotassium iodide 2 gDistilled water 100 ml Verhoeff’s solutionSolution a 20 mlSolution b 8 mlSolution c 8 mlAdd in the above order, mixing between

additions.

METHOD1. Deparaffinise sections and take to water.2. Stain in Verhoeff’s solution, 15–30 minutes.3. Rinse in water.4. Differentiate in 2% aqueous ferric chloride untilelastic tissue fibers appear black on a graybackground.5. Rinse in water.6. Rinse in 95% alcohol to remove any staining dueto iodine alone.

7. Counterstain as desired (van Gieson isconventional, although eosin may be used).8. Blot to remove excess stain.9. Dehydrate rapidly through ascending grades ofalcohol.10. Clear in xylene and mount in permanent

mounting medium.

Results:Elastic tissue fibers - blackOther tissues according to counterstain.

STAINING OF RETICULAR FIBRESTechniques for the demonstration of reticular

fibers may be divided into those using dyes as a means of staining, and the metal impregnation methods.

Dye techniques for reticular demonstration cannot be considered completely reliable.

Staining techniques do not readily differentiate between collagen and reticular fibers.

Metal impregnation techniques provide contrast, enabling even the finest fibers to be resolved.

Preparation of silver solution To 5 ml of 10% aqueous silver nitrate solution add

concentrated ammonia, drop by drop, until the precipitate first formed dissolves, taking care to avoid any excess of ammonia.

Add 5 ml of 3% sodium hydroxide solution. Re-dissolve the precipitate by the addition of concentrated

ammonia, drop by drop, until the solution retains a trace of opalescence.

If at this stage any excess of ammonia is present, indicated by the absence of opalescence, add a few drops of 10% silver nitrate solution, to produce a light precipitate.

Make up the volume to 50 ml with distilled water. Filter before use. Store in a dark bottle.

Gordon & Sweets’ method for reticular fibers

Method:1. Deparaffinize sections and take to water.2. Treat with 1% potassium permanganate solution,5 minutes.3. Rinse in tap water.4. Bleach in 1% oxalic acid solution.5. Rinse in tap water.6. Treat with 2.5% iron alum solution for at least 15minutes.7. Wash well in several changes of distilled water.8. Place in a Coplin jar of silver solution, 2 minutes.

9. Rinse in several changes of distilled water.10. Reduce in 10% aqueous formalin solution, 2minutes.11. Rinse in tap water.12. Tone in 0.2% gold chloride solution, 3 minutes.13. Rinse in tap water.14. Treat with 5% sodium thiosulfate solution, 3minutes.15. Rinse in tap water.16. Counterstain as desired.17. Dehydrate through ascending grades of alcohol.18. Clear in xylene and mount in permanent mountingmedium.

RESULTSReticular fibers - blackNuclei - black or unstainedOther elements according to counterstain

Preparation of silver solution:To 10 ml of 10% potassium hydroxide solution add 40

ml of 10% silver nitrate solution.Allow the precipitate to settle and decant the

supernatant. Wash the precipitate several times with distilled water. Add ammonia drop by drop until the precipitate has

just dissolved. Add further 10% silver nitrate solution until a little

precipitate remains. Dilute to 100 ml and filter.Store in a dark bottle.

Gomori’s method for reticular fibers

1. Deparaffinize sections and take to water.2. Treat with 1% potassium permanganate

solution,2 minutes.3. Rinse in tap water.4. Bleach in 2% potassium metabisulfate

solution.5. Rinse in tap water.6. Treat with 2% iron alum, 2 minutes.7. Wash in several changes of distilled water.

METHOD

8. Place in Coplin jar of silver solution, 1 minute.9. Wash in several changes of distilled water.10. Reduce in 4% aqueous formalin solution, 3minutes.11. Rinse in tap water.12. Tone in 0.2% gold chloride solution, 10 minutes.13. Rinse in tap water.14. Treat with 2% potassium metabisulfate solution,1 minute.15. Rinse in tap water.16. Treat with 2% sodium thiosulfate solution,1 minute.

17. Rinse in tap water.18. Counterstain as desired (van Gieson or eosin

issuitable).19. Dehydrate through ascending grades of

alcohol.20. Clear in xylene and mount in permanent

mounting medium.

Results:Reticular fibers - blackNuclei - grayOther tissues - according to counterstain

STAINS FOR CARBOHYDRATES

PERIODIC ACID SCHIFF TECHNIQUE Periodic acid solution:Periodic acid 1 gDistilled water 100 ml

Preparation of Schiff reagent:Dissolve 1 g of basic fuchsin and 1.9 g of sodium

metabisulfite in 100 ml of 0.15 M hydrochloric acid. Shake the solution at intervals or on a mechanical

shaker for 2 hours. The solution should be clear and yellow to light

brown in colour.

Add 500 mg of activated charcoal and shake for 1 to 2 minutes.

Filter the solution through a No. 1 Whatman filter into a bottle.

The filtered solution should be clear and colorless.

If the solution is yellow, repeat the charcoal decolorization using a fresh lot of activated charcoal.

Store at 4°C. Solution is stable for several months.

1. Dewax in xylene and rehydrate through graded

ethanol to distilled water.2. Oxidize with periodic acid for 5 minutes.3. Rinse in several changes of distilled water.4. Cover the sections with Schiff reagent for 15minutes.5. Rinse in running tap water for 5–10 minutes.6. Stain the nuclei with hematoxylin.

Differentiate and blue the sections.

METHOD

7. Dehydrate in graded ethanol and clear withxylene.8. Coverslip.

Results:Glycogen, neutral/ sialomucins - magentaVarious glycoproteins - magentaNuclei - blue

ALCIAN BLUE TECHNIQUE Alcian blue solution:Alcian blue 8GX 1 g3% acetic acid solution 100 ml Nuclear fast red:Aluminium sulphate Al2(SO4)3•18H2O 5 gDistilled water 100 mlNuclear fast red 0.1 g Dissolve the aluminium sulphate in the water

with heat. Add the nuclear fast red to water while still hot and filter.

1. Dewax in xylene and rehydrate through graded

ethanol to distilled water.2. Stain in the alcian blue solution for 30

minutes.3. Rinse in running tap water for 5 minutes.4. Counterstain in nuclear fast red for 10

minutes.5. Wash in running tap water for 1 minute.6. Dehydrate in graded ethanol.7. Clear in xylene and mount in a miscible

medium.

METHOD

Acid mucins (sulfomucins and sialomucins) - blue

Proteoglycans and hyaluronic acid - blueNuclei - red

RESULTS