Purpose of staining

1. To make various tissue components visible

2. To permit distinctions to be made between the tissue components

Mechanism:

1. Dyes behave like acidic or basic compounds which forms the basis of their selectivity
o Dyes form electrostatic linkages (salt) linkages with ionizable radicals of molecules in tissues
 Nucleic acids,GAG’s are anionic (negatively charged)- stain more readily with basic
dyes and are termed basophilic
 Toluidine blue, alcian blue and methylene blue
 hematoxylin
 Proteins, mitochondria, secretory granules and collagen are cationic (positively
charged)- have affinity for acidic dyes and are termed acidophilic
 Eosin, orange G and acidic fuchsin

Hematoxylin and Eosin (H&E)

o Hematoxylin reacts like a basic dye with a purplish blue color.

 It stains acidic, or basophilic, structure including the cell nucleous (which contains
DNA and nucleoprotein), and organelles that contain RNA such as ribosomes and
the rough endoplasmic reticulum.
o Eosin is an acidic dye that is typically reddish or pink. It stains basic, or acidophilic, structures
which includes the cytoplasm, cell walls, and extracellular fibers.

o In a clinical histology laboratory, all specimens are initially stained with H&E
o Special or advanced stains are only ordered if additional information is needed to provide a
more detailed analysis
 to differentiate between two morphologically similar cancer types.
o Routine use in histopathology laboratories
o provides the pathologist/researcher a very detailed view of the tissue
 clearly stains cell structures including the cytoplasm, nucleus, and organelles and
extra-cellular components
 information is often sufficient to allow a disease diagnosis based on:
 the organization (or disorganization) of the cells
 show abnormalities or particular indicators in the actual cells (such as
nuclear changes typically seen in cancer).
o Even when advanced staining methods are used, the H&E stain still forms a critical part of
the diagnostic picture as it displays the underlying tissue morphology which allows the
pathologist/researcher to correctly interpret the advanced stain.
2. Chemical basis of specialized stains use more complicated mechanisms

Periodic Acid Schiff (PAS)

o Feulgen reaction

 PAS stain is mainly used to highlight the molecules (structures) with high percentage
of carbohydrate content such as glycogen, glycoproteins, and proteoglycans
typically found in connective tissue, glycocalyx and basal laminae.

 Deoxyribose sugars are hydrolyzed by mild HCl which is then treated with PAS
reagent
 1,2glycol groups present in sugars are transformed into aldehyde residues
 Aldehyde residues then react with the Schiff reagent to produce a purple or
magenta color
 Enzyme digestion: retreatment of a tissue section with an enzyme that specifically
digests one substrate
 Ribonuclease pretreatment will greatly reduce cytoplasmic basophilia with
little overall effect on the nucleus
 Amylase pretreatment digests polysaccharides used to distinguish glycogen
from glycoproteins in PAS + material
 PAS staining can be used to assist in the diagnosis of several medical conditions such
as:
 Glycogen storage disease (vs. other storage disease)
 Adenocarcinoma which often secretes mucin
 Paget’s disease of breast
 Alveolar soft part sarcoma
 Staining macrophages in Whipple’s disease
 Erythroleukemia, Leukemia of immature RBCs
 Fungal infection (cell wall stain magenta)
Lipid Soluble dyes
 Indicated to lipid rich structures
 Pricessing steps that remove lipids should be avoided such as treatment with heat, organic
solvents or paraffin
 Frozen section is indicated
 Sudan black
o Lipophilic dye which dissolves lipid rich structures of cells
 Specialized methods to localize cholesterol, phospholipids and glycolipids are useful in the
diagnosis of metabolic diseases

Precautions in Staining:

1. Stains on the skin should be avoided. They are health hazards

 Effectively removed from the skin by prompt topical applicaton of 0.5% acid alcohol
followed by rinsing with tap water
  Many of the synthetic special stains are found to be genotoxic, mutagenic, immunotoxic and
carcinogenic
2. Failure of sections to remain on the slide during staining may be due to dirty or oily slide or albumin
fixative may be too old
3. If the section does not stain, the staining solution may be faulty. Impurities found on the dye or in
the water solvent will affect both the solubility and intensity of the dye
4. Failure of staining may be due to paraffin, fixative or decalcifying solution that has not been
thoroughly washed out and removed
5. Stains may be saved and used again for as long as they have not lost their staining properties
6. If, after staining sections do not appear clear under the microscope, xylol should be replenished
7. If the tissue is thoroughly adherent to the slide. It can be taken back several times for staining
without any danger of peeling off.