A comprehensive reference for histological, immunohistochemical and special staining methods used in diagnostic and research pathology laboratories worldwide.
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The foundational staining techniques used in every histopathology laboratory for general tissue examination and morphological assessment.
Principle: Haematoxylin, a basic dye, binds to acidic (basophilic) structures such as DNA and RNA in cell nuclei, staining them blue-purple. Eosin, an acidic dye, binds to basic (eosinophilic) structures such as cytoplasmic proteins, collagen and muscle fibres, staining them pink-red.
Nuclei: Blue to purple | Cytoplasm: Pink to red | Collagen: Pale pink | Red blood cells: Bright red/orange | Muscle fibres: Deep pink
Principle: Periodic acid oxidises vicinal glycol groups (1,2-diols) in carbohydrates to form dialdehydes. These aldehydes react with Schiff's reagent (leucofuchsin) to produce a magenta-coloured compound.
PAS-positive structures: Magenta/deep pink | Nuclei: Blue (haematoxylin counterstain) | Background: Pale pink to colourless
Diastase control (PAS-D): Pre-treatment with diastase (amylase) removes glycogen. Structures that are PAS-positive but diastase-resistant include basement membranes, mucins and fungi.
Principle: A Romanowsky-type stain composed of methylene blue, eosin and azure dyes. The differential binding of acidic and basic dye components produces polychromatic staining of blood and tissue elements.
Nuclei/chromatin: Dark blue-purple | Cytoplasm (basophilic): Blue | Cytoplasm (eosinophilic): Pink-red | Bacteria (H. pylori): Dark blue | Mast cell granules: Purple-red
Principle: A polychromatic Romanowsky stain containing methylene blue and eosin in methanol. Widely used for peripheral blood smear examination and differential white blood cell counts.
Neutrophil granules: Lilac/pink | Eosinophil granules: Bright red-orange | Basophil granules: Dark blue-black | Lymphocyte cytoplasm: Sky blue | Red blood cells: Pink-orange
Histochemical techniques that selectively demonstrate specific tissue components, organisms or deposits not readily identifiable on routine H&E staining.
Target: Collagen fibres
Principle: Differential staining of muscle, collagen and nuclei using three dyes — Weigert's iron haematoxylin, Biebrich scarlet-acid fuchsin, and aniline blue (or light green).
Results: Collagen — blue/green | Muscle & cytoplasm — red | Nuclei — black
Use: Liver fibrosis staging, cardiac fibrosis, renal fibrosis assessment.
Target: Reticular fibres (type III collagen)
Principle: Silver impregnation technique. Reticular fibres reduce ammoniacal silver to metallic silver, which is then toned with gold chloride.
Results: Reticular fibres — black | Background — grey to pink
Use: Liver architecture assessment, lymph node framework, bone marrow fibrosis grading.
Target: Amyloid deposits
Principle: Congo red dye binds to the beta-pleated sheet structure of amyloid fibrils in a highly ordered manner.
Results: Amyloid — salmon pink/red (brightfield); apple-green birefringence under polarised light | Nuclei — blue (haematoxylin counterstain)
Use: Diagnosis of systemic and localised amyloidosis.
Target: Acid-fast bacilli (Mycobacterium tuberculosis, atypical mycobacteria)
Principle: Carbol fuchsin penetrates the waxy mycolic acid cell wall when heated. Acid-alcohol decolourises non-acid-fast organisms, but mycobacteria retain the dye.
Results: Acid-fast bacilli — bright red | Background — blue (methylene blue counterstain)
Use: Tuberculosis diagnosis, leprosy (modified ZN).
Target: Fungal cell walls (Pneumocystis, Aspergillus, Candida, Histoplasma)
Principle: Chromic acid oxidises polysaccharides in fungal cell walls to aldehydes. These reduce methenamine silver to metallic silver, which deposits on fungal structures.
Results: Fungi — black/dark brown | Background — pale green (light green counterstain)
Use: Detection of fungal infections in tissue sections.
Target: Haemosiderin (ferric iron, Fe³⁺)
Principle: Ferric iron reacts with potassium ferrocyanide in the presence of hydrochloric acid to form ferric ferrocyanide (Prussian blue), an insoluble blue pigment.
Results: Iron deposits — bright blue | Nuclei — red (neutral red counterstain)
Use: Iron overload assessment, haemochromatosis, sideroblastic anaemia, transfusion haemosiderosis.
Target: Acid mucopolysaccharides (acid mucins, glycosaminoglycans)
Principle: Alcian blue, a cationic (basic) dye, binds to negatively charged carboxyl and sulphate groups of acid mucins at pH 2.5. At pH 1.0, only strongly acidic sulphated mucins stain.
Results: Acid mucins — blue/turquoise | Nuclei — red (neutral red counterstain)
Use: Identification of acid mucins, Barrett's oesophagus (AB/PAS combination), mesothelioma.
Target: Epithelial mucin
Principle: Carmine dye in an aluminium-containing solution selectively binds to the acidic groups of epithelial mucin.
Results: Epithelial mucin — deep red/magenta | Capsule of Cryptococcus neoformans — red/magenta | Nuclei — blue/black
Use: Mucin-secreting adenocarcinomas, cryptococcal infection identification.
Target: Neutral lipids, triglycerides, fatty acids
Principle: Oil Red O is a lysochrome (fat-soluble dye) that partitions into lipid droplets, being more soluble in lipid than in the solvent carrier. Requires frozen sections as lipids are dissolved by processing solvents.
Results: Lipids — bright red/orange-red | Nuclei — blue (haematoxylin counterstain)
Use: Fatty liver assessment, lipid storage diseases, fat embolism. Frozen sections only.
Target: Elastic fibres and collagen
Principle: Verhoeff's elastic stain (iron haematoxylin) demonstrates elastic fibres. Van Gieson's counterstain (picric acid + acid fuchsin) differentiates collagen from muscle and other tissues.
Results: Elastic fibres — blue-black/black | Collagen — red | Muscle & cytoplasm — yellow
Use: Vascular pathology, temporal arteritis, emphysema, skin elastic tissue assessment.
Target: Melanin, argentaffin granules
Principle: Argentaffin reaction — melanin granules and enterochromaffin cells reduce ammoniacal silver without an external reducing agent, depositing metallic silver on the target.
Results: Melanin — black/dark brown | Argentaffin granules — black | Nuclei — red (nuclear fast red counterstain)
Use: Confirming melanin in pigmented lesions, carcinoid tumour identification.
Target: Calcium deposits (calcium phosphate, calcium carbonate)
Principle: Silver nitrate reacts with the phosphate or carbonate anions associated with calcium deposits. Exposure to strong light reduces the silver salt to metallic silver.
Results: Calcium deposits — black/dark brown | Nuclei — red (nuclear fast red counterstain)
Use: Pathological calcification, calcinosis cutis, calcium oxalate identification (with polarised light).
Immunohistochemistry uses antibodies to detect specific antigens in tissue sections, providing critical information for tumour classification, prognostic assessment and treatment selection.
Formalin fixation causes protein cross-linking that can mask antigenic epitopes. Antigen retrieval reverses this cross-linking to restore antibody binding.
Molecular pathology techniques complement morphology and immunohistochemistry, providing genetic and genomic data essential for precision medicine and targeted therapy selection.
Principle: Fluorescently labelled DNA probes hybridise to complementary sequences on chromosomes in tissue sections or cell preparations. Signals are visualised using fluorescence microscopy.
Key applications:
Principle: Polymerase chain reaction amplifies specific DNA sequences exponentially through repeated cycles of denaturation, annealing and extension. RT-PCR first reverse-transcribes RNA to cDNA.
Key applications:
Principle: Massively parallel sequencing enables simultaneous analysis of multiple genes, whole exomes or whole genomes from FFPE tissue or liquid biopsy samples.
Sample preparation overview:
Principle: Labelled nucleic acid probes (DNA or RNA) hybridise to complementary sequences within intact tissue sections, preserving morphological context. Chromogenic ISH (CISH) uses enzyme-labelled probes for brightfield microscopy.
Key applications:
The systematic preparation of tissue specimens from receipt in the laboratory through to the production of stained microscope slides for diagnostic examination.
Robust quality assurance and quality control programmes are essential to ensure reliable, reproducible and clinically accurate results in histopathology and immunohistochemistry laboratories.
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