Principles of Staining

Histopathology Report on the Principles of Staining

1. PRINCIPLES OF STAINING McLean, Charlotte Ong , Domingo

2. Objectives: To differentiate the different staining techniques To reveal the presence of various internal and external structures To produce specific chemical and physical reaction

3. STAINING is the process of applying dyes on the sections to see and study the architectural pattern of the tissue and physical characteristics of the cell.

4. Alcian Blue and PAS (intestine). A stain that combines the properties of both Alcian Blue and Periodic Acid Schiff staining. On a chemical basis, certain parts of cells and tissues that are acidic in character ( e.g nucleus) have greater affinity for basic dyes, while basic constituents ( e.g cytoplasm) take more of the acid stains In general, microscopic examination is facilitated if two contrasting stains are used; e.g hematoxylin which stains the nuclear detail, and eosin which brings out the cytoplasmic detail of the cell and the tissues architecture

5. Gomori Trichrome Blue is used to stain and identify muscle fibers, collagen and nuclei 3 Major Groups of Staining 1. Histological Staining Process whereby the tissue constituents are demonstrated in sections by direct interaction with a dye or staining solution, producing coloration of the active tissue component . - micro anatomical or histologic staining is used to demonstrate the general relationship of tissues and cells with differentiation of nucleus and cytoplasm

6. 2. Histochemical Staining (HISTOCHEMISTRY) Various constituents of tissues are studied thru chemical reactions that will permit microscopic localization of a specific tissue substance. Example: - Perls prussian blue reaction for hemoglobin Periodic Acid Schiff staining for Carbohydrates Perls prussian blue

7. Confocal fluorescence micrograph of HeLa cells stained with monoclonal antibody against mitochondria enzyme and Cy3-conjugated anti-mouse antibody (red); rabbit polyclonal antibody to histones in DNA and Cy5-conjugated-rabbit antibody (blue). 3. Immunohistochemical Staining - A combination of immunologic and histochemical techniques that allow phenotypic markers to be detected and demonstrated under the microscope, using a wide range of polyclonal or monoclonal fluorescent labeled or enzyme-labeled antibodies

8. Human cheek cells stained with methylene blue Prostate section stained with Hematoxylin & Eosin (cytoplasm, pink; nuclei,blue ) Methods Of Staining DIRECT STAINING - A process of giving color to the sections by using aqueous or alcoholic dye solution - e.g methylene blue, eosin

9. INDIRECT STAINING - A process whereby the action of the dye is intensified by adding another agent; either mordant or accentuator "MORDANT" - serves as a link or bridge between the tissue and the dye, to make the stainig resction possible. - e.g Potassium alum with hematoxylin in Erlichs hematoxylin , and iron in Weigers hematoxylin . "ACCENTUATOR" - Does not participate in the staining reaction, but merely accelerates or hastens the speed of the staining reaction by increasing the staining power and selectivity of the dye. - e.g Lofflers methylene blue and phenol in carbol thionine and carbol fuchsin

10. This is a section of bone marrow stained with Weigert's haematoxylin and counterstained with eosin Modified Loeffler's methylene blue staining of Rhodococcus jostii

11. REGRESSIVE STAINING - Tissue is overstained to o bliterate the Cellular details, and the excess stain is removed or decolorized from unwanted parts of the tissue, until desired intensity of color is obtained. Lung tissue stained with the H&E technique. Nuclei are darkly stained in this image

12. PROGRESSIVE STAINING A process whereby tissue elements are stained in definite sequence, and staining solution is applied for specific periods of time or until the desired intensity of coloring of the different tissue elements is attained. It result to "diffused color and obscured details" due to difficulty of producing sufficiently intense progressive staining of cell structure without staining other parts - less favoured than regressive staining

13. DIFFERENTIATION (DECOLORIZATION) selective removal of excess stain from the tissue during regressive staining in order that a specific substance may be stained distinctly from the surrounding tissues . usually done by washing the section in simple solution ( e.g water or alcohol) or by use of acid and oxidizing agents.

14. Use of specific dyes which differentiate particular substances by staining t-hem with a color that is different from that of the stain itself ( metachromasia ) for cartilage, conmective tissue, epithelial mucin , mast cell granules, and amyloid . metachromatic dyes are basic dyes belonging to the " thizine and triphenylmethane " groups, such as: methyl violet or crystal violet cresyl blue safranin Bismarck brown Basic fuchsin Methylene blue Thionine toluidine blue Azure A, B, C METACHROMATIC STAINING

15. All metachromatic dyes are cations or basic whose peculiar staining property dpends upon their tendency to polymerize . All tissue components shown metachromasia are large anionic or acidic molecules containing large amount of sulfate , phosphate, or carboxylic acid radicals . WATER is necessary for most metachromatic staining techniques, and metachromasia is usually lost if the section is dehydrated in ALCOHOL after staining . the major group of metachromatic tissues consists of acidic polysaccharides that occur in ground substance of cartilage ( chrondoitin sulfate ) and in connectiv e tissue mucin (acid mucopolysaccharide ) that bind basic dyes.

16. COUNTERSTAINING - Application of different color or stain to provide contrast and background to the staining of the structural components to be demonstrated. CYTOPLASMIC STAINS NUCLEAR STAINS red yellow green Eosin Y Picric acid Light green SF Eosin B Orange G Lissamine green Phloxine B Rose bengal RED BLUE neutral red Methylene blue Safranin O Toluidine blue Carmine Hematoxylin Celestine blue

17. METALLIC IMPREGNATION Is a process where specific tissue elements are demonstrated , not by stains, but by colourless solutions of metallic salts which are thereby reduced by the tissue, producing an opaque usually black deposit on the surface of the tissue or bacteria. a metallic impregnating agent is different from a stain in that it is not absorbed by the tissue, but is held physically on the surface as a precipitate or as a reduction product in certain tissue components . It is utilized for silver staining of nervous system and also used to demonstrate reticulin . e.g. gold (gold chloride)and Silver nitrate, most commonly used agent for impregantion , can also function as staining agent . ammoniacal silver is reduced by argentaffin cells (in melanin and intestinal glands), that forms black deposit seen under the microscope.

18. VITAL STAINING - Selective staining of living cell constituents, demonstrating cytoplasmic structure by phagocytosis of the dye particle ( cytoplasmic phagocytosis ). a. INTRAVITAL STAINING - Staining of living cell is done by injecting the dye inti any part of animal body (either intravenous, intraperitoneal or subcutaneous), producig specific coloration of certain cells, particularly those of the reticulo -endothelial system. lithium , carmine and india ink

19. SUPRAVITAL STAINING - used to stain living cells immediately after removal from the living body. common dyes are: Neutral red – best vital stain Janus green – recommended for mitochondria Trypan blue – 1 g of dye is dissolved in 100ml of sterile dis. Water to be used immediately; dangerous to stand for more than 1 hr, for it is toxic to the cell. Nile blue Thionine Toluidine blue

20. STAINING OF PARAFFIN SECTIONS

21. MATERIALS USED FOR STAINING Coplin jar – slotted jar holding from 5-9 slides. Slotted staining dishes – holding from 5-19 slides, over which different solutions are poured. Slides are placed on end singly or in staggered fashion, in the arm. Metal or glass staining racks or carriers – holding from 10-30 slides upright.

22. 4 Staining M ethods C ommonly E mployed for F rozen S ections a. Hematoxylin -Eosin Method b. Thionine Method c. Polychrome Methylene Blue Method d. Alcoholic Pinacyanol Method ( used also for suparavital staining of MITOCHONDRIA and primarily for color sensitization in photography)

23. Routine H&E staining in Paraffin Embedded Section (Regressive Staining) Routine Hematoxylin and Eosin (H&E) staining- the most common method utilized for microanatomical studies of tissues Using regressive staining which consists of overstating the nuclei, removal of superfluous and excessive color of the tissue constituent by 'acid differentiation'. Most fixatives can be used EXCEPT OSMIC ACID SOLUTIONS which inhibit hematoxylin

24. RESULTS: nuclei – blue to blue black Cytoplasm, proteins in edema fluid – pale pink Karyosome – dark blue RBCs , eosinophilic granules, keratin – bright orange-red Basophil cytoplasm, plasma cells & osteoblast -purplish pink Cartilage – pink or light blue to dark blue (depends on the type of stain used, darkest with Erlichs hematoxylin ) Calcium and calcified bone – purplish blue Decalcified bone matrix, collagen and osteoid – pink Muscle fibers – deep pink

25. H&E staining of frozen section for Rapid diagnosis ( Progressive staining) Reagents for this rapid H&E stain are generally arranged in sequence using a series of coplin jars. This method takes only 5-10 min Produces well differentiated sections that are semi- permanet and can be stored. The remaning portion of tissue must be kept for routine procassing and are made for comparison with frozen sections.

26. RESULTS: Cells nuclei, cytoplasmic inclusions and muscle striations stain black. Other constituents are colored according to counterstain . Heidenhains Iron Hematoxylin Method Hamster Esophagus fixed in Bouin's Fluid and stained with Heidenhain's Hematoxylin only, at 40x

27. Celestine Blue- Haemalum Sequence Staining Celestine blue is an oxazine dye used as an alternative to iron hematoxylin nuclear stain, producing a strong and precise nuclear stain that is resistant to decolorization by succeeding acid stains and solutions . - forms a strong staining lake with 'iron alum', acting as a mordant to bind hematoxylin RESULT : Cell nuclei – blue Other constituents colored according to the counterstain used a section of bone marrow, from the same block, stained with the Celestin blue - haemalum method and counterstained with eosin.

28. Mallorys Phloxine Methylene Blue Stain Known as Eosin- Methylene Blue Method (EMB) method Produce sharp nuclear stain Reveals marked differentiation the various structures in the tissues, which should be fixed in Zenkers fluid.

29. COLLOIDIONIZATION OF SECTIONS Collodionization Is the process of more firmly attaching of ribbons by coating the slide with dilute (thin) celloidin solutions . recommended for sections that will be subjected to strong alkaline or acid solutions and for tissues that contain glycogen for demonstration . - celloidin will be removed in the final dehydration with absolute alcohol prior to clearing and mounting.

30. STAINING OF CELLOIDIN SECTIONS C ellulose N itrate ( celloidin ) is soluble in absolute alcohol hence, treatment should be avoided with abs. Alcohol during dehydration and clearing of stained sections . - sections treated with 95% alcohol may be transferred to a mixture of equal parts of chlroform , absolute alcohol and xylene then treated with xylene and mounted to xam .

31. RE-STAINING OF OLD SECTIONS Immersed in xylene for 24 hrs or gently heated until mounting medium begins to bubble Coverslip may then removed by lifting it with a dissecting needle. Section is then placed in xylene for 30 min to remove the remaining balsam and then brought down to water . It is placed in a 0.5 potassium permanganate solution for 5-10 min, rinsed in tap water and subsequently immersed in 5% oxalic acid for 5 min or until the section is decolorized. Wash it again in running tap water for another 5 min - The section may then be restained with the appropriate staining technique.

32. BROKEN SLIDES for immediate examination: mounting a broken slide to another clean xylene -moist slide with a drop of mounting media ( Clarite or Permount ) may be sufficient . - replacement of slide is not available, the section (if still intact) may be transferred to another slide. - the coverslip can be removed by soaking in xylene , and placing the broken slide in the incubator at 37C until all the mountant has been removed. the whole slide is then covered with a mixture of 6 parts butyl acetate and 1 part durofix and left in the incubator for 30 min until the mixture hardens into a film . using a sharp scalpel blade, the hardened film is cut around the section, and the slide is placed in cold water until the film and section float off. - The film containing section is mounted in a clean glass slide, placed in the 37C incubator until dry, washed gently with butyl acetate, then washed well with xylene , and mounted in Clarite or Permount .