Special stains by sowmya

Special stains

1. -Dr Sowmya Srinivas SPECIAL STAINS

2. CONNECTIVE TISSUE STAINS CARBOHYDRATE STAINS LIPID STAINS NUCLEIC ACIDS STAINS PROTEINS MICRO ORGANISMS TYPES OF SPECIAL STAINS

3. 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

4. The connective tissues are divided into the following groups: Connective tissue proper – includes loose or areolar, dense, adipose, reticular Cartilage – hyaline elastic and fibrocartilage Bone – spongy or cancellous and dense or cortical Blood Blood-forming – hematopoietic.

5. Connective tissue includes- Collagen Elast in Reticul in Basement membrane Muscle

6. 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.

7. Collagen may be differentially stained by any of the following: Van Gieson's stain Masson's trichrome stain Mallory's trichrome stain Aniline blue stain Eosin Reticulin stain COLLAGEN STAINS

8. 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

9. Solution: Saturated aqueous solution of picric acid (approximately 1 %)- 100 ml 1 % 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.

10. STAINING TECHNIQUE Deparaffinise sections and bring to water. Stain with Weigert’s hematoxylin for 40 minutes. Wash in tap water. Differentiate in Acid alcohol. Wash well in tap water. Stain in Van Gieson stain for 3 minutes Blot and dehydrate through alcohols. Clear in Xylene and mount in permanent mounting medium.

11. RESULTS: Nuclei – Blue/black Collagen – Red Other 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.

12. MASSON TRICHROME STAIN Fixation: Formal saline. Sections: All types. Solution A: Acid Fuchsin – 0.5g Glacial acetic acid – 0.5ml Distilled water – 100ml

13. Solution B: Phosphomolybdic acid – 1g Distilled water – 100ml Solution C: Methyl blue – 2g Glacial acetic acid – 2.5ml Distilled water – 100ml.

14. Deparaffinise sections and take to water. Remove mercury pigment by iodine, sodium thiosulphate sequence. Wash in tap water. Stain nuclei by the celestine blue-hematoxylin method. Differentiate with 1% acid alcohol. Wash well in tap water. Stain in acid fuchsin Solution A for 5 minutes. Rinse in distilled water. METHOD

15. 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.

16. Nuclei – Blue/black Cytoplasm, muscle and RBC’s – Red Collagen – Blue. RESULTS

17. Staining of Elastic tissue fibres Verhoeff's method O rcein technique Weigert’s R esorcin-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

18. Preparation of stain: Solution a Hematoxylin 5 g Absolute alcohol 100 ml Solution b Ferric chloride 10 g Distilled water 100 ml Verhoeff’s method for elastic fibers

19. Solution c, Lugol’s iodine solution Iodine 1 g Potassium iodide 2 g Distilled water 100 ml Verhoeff’s solution Solution a 20 ml Solution b 8 ml Solution c 8 ml Add in the above order, mixing between additions .

20. METHOD 1. 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 until elastic tissue fibers appear black on a gray background. 5. Rinse in water. 6. Rinse in 95% alcohol to remove any staining due to iodine alone.

21. 7. Counterstain as desired (van Gieson is conventional, although eosin may be used). 8. Blot to remove excess stain. 9. Dehydrate rapidly through ascending grades of alcohol. 10. Clear in xylene and mount in permanent mounting medium. Results: Elastic tissue fibers - black Other tissues according to counterstain.

22. STAINING OF RETICULAR FIBRES Techniques 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.

23. 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

24. 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 15 minutes. 7. Wash well in several changes of distilled water. 8. Place in a Coplin jar of silver solution, 2 minutes.

25. 9. Rinse in several changes of distilled water. 10. Reduce in 10% aqueous formalin solution, 2 minutes. 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, 3 minutes. 15. Rinse in tap water. 16. Counterstain as desired. 17. Dehydrate through ascending grades of alcohol. 18. Clear in xylene and mount in permanent mounting medium.

26. RESULTS Reticular fibers - black Nuclei - black or unstained Other elements according to counterstain

27. 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

28. 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

29. 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, 3 minutes. 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 .

30. 17. Rinse in tap water. 18. Counterstain as desired (van Gieson or eosin is suitable). 19. Dehydrate through ascending grades of alcohol. 20. Clear in xylene and mount in permanent mounting medium. Results : Reticular fibers - black Nuclei - gray Other tissues - according to counterstain

31. STAINS FOR CARBOHYDRATES

32. PERIODIC ACID SCHIFF TECHNIQUE Periodic acid solution: Periodic acid 1 g Distilled 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.

33. 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.

34. 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 15 minutes. 5. Rinse in running tap water for 5–10 minutes. 6. Stain the nuclei with hematoxylin. Differentiate and blue the sections. METHOD

35. 7. Dehydrate in graded ethanol and clear with xylene. 8. Coverslip . Results : Glycogen, neutral/ sialomucins - magenta Various glycoproteins - magenta Nuclei - blue

36. ALCIAN BLUE TECHNIQUE Alcian blue solution: Alcian blue 8GX 1 g 3% acetic acid solution 100 ml Nuclear fast red: Aluminium sulphate Al2(SO4)3•18H2O 5 g Distilled water 100 ml Nuclear 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 .

37. 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

38. Acid mucins (sulfomucins and sialomucins ) - blue Proteoglycans and hyaluronic acid - blue Nuclei - red RESULTS