Staining Methods

1. Staining Methods

2. Steps in the processing of tissues 1. Fixation – preservation of tissues in its original condition. 2. Dehydration – removal of water from tissues. 3. Clearing – infiltration of paraffin solvent. 4. Embedding – infiltration of paraffin wax. 5. Microtomy– preparing thin slices of tissues. 6. Staining – colouring of tissues. 7. Mounting – arranging tissues on slides.

3. Why We Need To Stain the Tissues ???

4. Staining • Without staining, the tissue section would remain translucent and we would still have difficulty identifying the relevant cell types and features. • Staining using colored dyes provides a mechanism for introducing contrast.

5. Staining • stain tissue components more or less selectively, with many behaving like acidic or basic compounds.

6. Staining • Cell components such as nucleic acids with a net negative charge (anionic) stain more readily with basic dyes and are termed basophilic • cationic components, such as proteins with many ionized amino groups, have affinity for acidic dyes and are termed acidophilic. • Examples of basic dyes are toluidine blue, alcian blue, and methylene blue. • Hematoxylin , staining behaves like a basic dye basophilic tissue components.

7. Basophilic Acidophilic • Basophilic • Stain with basic dye [dye+Cl-] • Toluidine blue, methylene blue, hematoxylin, alcian blue • Nucleic acids, some cytoplasmic components (rRNA and rER), glycosaminoglycans and acidic glycoproteins • Stain with acidic dye [Na+dye-] • Orange G, eosin, acid fuschin • Mitochondria, cytoplasm, secretory granules, ECM proteins Tissue Staining

8. H&E Stain(Eeosinis the counterstain to Hematoxylin) • Hematoxylinproduces a dark blue or purple color, staining DNA in the cell nucleus and other acidic structures (such as RNA-rich portions of the cytoplasm and the matrix of cartilage). • In contrast, Eosinstains other cytoplasmic components and collagen. In many staining procedures certain structures such as nuclei become visible, but other parts of cells remain color free.

9. H&E Stain

10. Other notable stains • Masson Trichrome– most common • Usually used to look at collagen in the extracellular matrix • Nuclei (basophilic) – blue/black • Collagen – green orblue • Cytoplasm, muscle, keratin, erythrocytes – red

11. Masson Trichrome

12. Silver stain • Silver stains reticular and collagen fibres in brown to black. • Silver methods are used for staining of neurons in neurohistology.

13. Gold and Silver Stains Silver stain of reticular fibers (type III collagen) String-like appearance in this image Gold staining of motor end plate in muscle