Staining imparts contrast to translucent tissue sections, allowing cellular and extracellular components to be distinguished under brightfield microscopy. The standard stain in histopathology is hematoxylin and eosin (H&E), but dozens of specialized stains exist to highlight specific structures, microorganisms, and chemical substances.
Hematoxylin and Eosin (H&E)
H&E is the workhorse stain of histopathology, used on virtually every routinely processed section. Hematoxylin, a basic dye, binds acidic structures (nuclei, ribosomes, rough endoplasmic reticulum) and stains them blue-purple (basophilic). Eosin, an acidic dye, binds basic structures (cytoplasmic proteins, collagen, red blood cells) and stains them pink-red (eosinophilic).
An adequately stained H&E section shows crisp nuclear detail with open or condensed chromatin, visible nucleoli, and well-defined nuclear membranes. The cytoplasm should display appropriate tinctorial qualities — pancreatic acinar cells appear granular due to zymogen granules, muscle cells appear eosinophilic and fibrillar, and cartilage matrix appears glassy and basophilic. Artifacts such as over-staining (muddy nuclei), under-staining (pale nuclei), or uneven staining indicate problems in the staining process or poor tissue processing.
Special Stains for Connective Tissue
Masson’s Trichrome stains collagen blue or green, muscle and cytoplasm red, and nuclei black. It is used to evaluate fibrosis in liver disease, muscle dystrophies, and myocardial scarring.
Verhoeff-van Gieson (VVG) stains elastic fibers black, collagen red, and muscle yellow. It is essential for assessing vascular diseases and elastic fiber disorders.
Reticulin stain (silver impregnation) stains type III collagen (reticulin fibers) black. It highlights the architectural framework of organs and is critical for diagnosing bone marrow disorders and liver diseases.
Periodic Acid-Schiff (PAS) stains glycogen, glycoproteins, and fungal cell walls magenta. It is used to identify fungal organisms, diagnose glycogen storage diseases, and highlight basement membranes in renal biopsies. PAS with diastase (PAS-D) digestion distinguishes glycogen (removed by diastase) from other PAS-positive material.
Stains for Microorganisms
Gram stain (tissue adaptation) differentiates Gram-positive (purple) from Gram-negative (pink) bacteria in tissue sections. Unlike the bacterial Gram stain technique used in microbiology, the tissue Gram stain works on paraffin sections.
Ziehl-Neelsen stains acid-fast bacilli (Mycobacterium tuberculosis) bright red against a blue or green counterstain.
Grocott’s methenamine silver (GMS) stains fungal cell walls black and is the most sensitive stain for detecting fungi in tissue.
Warthin-Starry stains spirochetes and Helicobacter pylori black on a pale yellow background.
Stains for Cytoplasmic Granules and Inclusions
Giemsa stain is used for bone marrow trephine biopsies to evaluate hematopoietic cells, and can detect Helicobacter pylori, mast cells, and certain parasites.
Luxol Fast Blue (LFB) stains myelin blue-green and is used in neuropathology to assess demyelinating diseases.
Congo Red stains amyloid deposits salmon-pink under brightfield illumination and shows apple-green birefringence under polarized light — the diagnostic standard for amyloidosis.
Histochemistry and Enzyme Histochemistry
Histochemistry uses chemical reactions to localize specific substances in tissue sections. Perls’ Prussian Blue detects ferric iron (hemosiderin) as a blue precipitate — used to diagnose iron overload disorders. Von Kossa stain detects calcium deposits (black or brown) in soft tissues. Oil Red O (on frozen sections) stains neutral lipids orange-red.
Enzyme histochemistry detects enzymatic activity in situ. Examples include alkaline phosphatase (bone and liver), acid phosphatase (prostate), and non-specific esterase (macrophages). These techniques require fresh or frozen tissue because fixation inactivates enzymes.
Immunohistochemistry
Immunohistochemistry (IHC) uses antibodies conjugated to enzymes (horseradish peroxidase, alkaline phosphatase) to detect specific antigens in tissue sections. The reaction is visualized with chromogens such as DAB (brown) or Fast Red (red). IHC has largely replaced many special stains and enzyme histochemistry techniques because of its superior specificity. It is indispensable for tumor classification (keratins, CD markers), infectious disease diagnosis, and predictive biomarker testing (HER2, PD-L1, mismatch repair proteins).
