Staining Methods

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.

Why We Need To Stain the Tissues ???

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.

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

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.

Tissue Staining Basophilic Acidophilic Stain with acidic dye [Na+dye-] 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

H&E Stain (Eeosin is the counterstain to Hematoxylin) Hematoxylin produces 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, Eosin stains other cytoplasmic components and collagen. In many staining procedures certain structures such as nuclei become visible, but other parts of cells remain color free.

H&E Stain

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

Masson Trichrome

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

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