1. HE staining
Materials: Paraffin sections
Dyes: Hematoxylin stain is alkaline, mainly staining the chromatin in the cell nucleus and the ribosomes in the cytoplasm purple-blue; eosin is an acidic dye, mainly staining the components in the cytoplasm and extracellular matrix red.
Application: General tissue changes and tissue products can be displayed by this staining method, which is the most commonly used staining method in morphology.
Results: The cell nucleus is stained bright blue by hematoxylin, the cartilage matrix and calcium salt particles are dark blue, and the mucus is gray-blue. The cytoplasm is stained with different shades of pink to pink by eosin, and the eosinophilic granules in the cytoplasm are bright red with strong reflection. Collagen fibers are light pink, elastic fibers are bright pink, red blood cells are orange-red, and proteinaceous fluid is pink.
2. Toluidine blue staining
Materials: paraffin sections, cell slides
Dyes: Toluidine blue, a synthetic alkaline dye, acidic substances in tissue cells are stained by combining with cations
Applications: initial screening of condyloma acuminatum and detection of mast cells, staining of intracellular acidic mucopolysaccharides, and staining of nerve cell Nissl bodies
Results: The nucleus can be stained blue, and heterochromatic substances such as heparin and histamine in the cytoplasm of mast cells can be easily stained purple-red, cartilage and osteoblasts are purple-red, acidic mucopolysaccharides are red, and Nissl bodies are dark blue.
3. Sirius red picric acid staining
Materials: 4% formaldehyde-fixed tissue paraffin sections
Dye: Sirius red-saturated picric acid solution
Application: Using the different characteristics of collagen polymerization and its winding spiral arrangement, this staining method can display four types of collagen fibers under polarized light microscope according to the difference in birefringence and coloring.
Results: Ordinary light microscope: collagen fibers are red, cell nuclei are green, and other components are yellow.
Polarized light microscope: Type I collagen fibers: red or yellow, tightly arranged, with strong birefringence;
Type II collagen fibers: multiple colors, loose reticular, weak birefringence;
Type III collagen fibers: green, thin fibers, weak birefringence;
Type IV collagen fibers: light yellow, weak birefringence.
4. TUNEL Staining
Materials: paraffin-embedded, frozen and ultrathin sections, cells
Dye: Fluorescein-labeled dUTP can be linked to the 3'-OH end of the broken DNA in apoptotic cells under the action of terminal deoxyribonucleotidyl transferase (TdT enzyme), and specifically bind to the fluorescein antibody linked to horseradish peroxidase (HRP), which in turn reacts with the HRP substrate diaminobenzidine (DAB) to produce a strong color reaction.
Application and results: In situ detection of apoptosis at the single-cell level, efficacy evaluation of anti-tumor drugs, and determination of cell death type and differentiation stage by two-color method. The nuclei of apoptotic cells are stained dark brown, while normal or proliferating cells are not stained.
5. Immunostaining
Including immunohistochemistry (IHC)/immunocytochemistry (ICC) and immunofluorescence (IF), all of which are qualitative, localized and relatively quantitative studies of antigens (peptides and proteins) in tissue cells by visualizing antigen-antibody binding reactions. The colorants used in IHC and ICC are mostly enzymes, that is, chemical color development; the colorants used in IF are mostly fluorescein, that is, fluorescent color development.
Materials: cells, frozen sections, paraffin sections
Applications and results: diagnosis and differential diagnosis of malignant tumors, confirmation of the primary site of metastatic malignant tumors, pathological classification of tumors, confirmation of micrometastases, localization and relative quantification of positive products, and detection of cell apoptosis.
References
Parra, E.R., Uraoka, N., Jiang, M. et al. Validation of multiplex immunofluorescence panels using multispectral microscopy for immune-profiling of formalin-fixed and paraffin-embedded human tumor tissues. Sci Rep 7, 13380 (2017).
