Tyramide Amplification Buffer

Ki-67 immunostaining measures the growth fraction of a cell population by detecting Ki-67, a nuclear antigen present in proliferating cells and absent in quiescent G0 cells. The readout is the percentage of Ki-67-positive nuclei among total counted cells, commonly called the Ki-67 labeling index or proliferation index.

Polymer-based two-step IHC detects tissue antigens by first binding an unlabeled primary antibody to the antigen and then applying an HRP-polymer secondary reagent that carries multiple secondary antibodies and HRP molecules on a polymer backbone; the localized HRP converts chromogens such as DAB or AEC into visible deposits for light-microscopic interpretation. The method is \"two-step\" because the primary antibody step is followed directly by the polymer-enzyme secondary reagent, rather than by separate secondary-antibody and avidin-biotin complex steps; published comparisons reported similar or higher sensitivity than several multistep systems and avoidance of endogenous-biotin interference.

Immunohistochemistry-Frozen (IHC-F) of frozen samples is a widely used technique for detecting and locating specific antigens within preserved cellular structures. Unlike formalin-fixed paraffin-embedded samples, frozen tissues retain their natural antigenicity, making them particularly suitable for targets sensitive to chemical fixation, and the procedure is relatively simple and rapid.

Avidin-biotin or streptavidin-biotin immunohistochemistry detects tissue antigens by binding a primary antibody to the target antigen, then detecting that antibody with a biotinylated antibody and an avidin-biotin-enzyme or streptavidin-enzyme detection complex; the enzyme reaction produces a visible chromogenic deposit at the antigen site for light-microscopic localization. The classic ABC method uses the high-affinity avidin-biotin interaction to bridge biotinylated secondary antibody and biotinylated peroxidase, and early comparative studies reported stronger immunoperoxidase staining than PAP-based methods in formalin-fixed tissue sections.

Double or multiplex chromogenic IHC detects two or more protein targets in the same FFPE tissue section by repeated antigen-antibody binding, enzyme-linked detection, chromogen deposition, image capture, and, for higher-plex workflows, removal or destaining before the next staining cycle. Chromogenic readouts are generated as colored precipitates at antigen sites, enabling evaluation of marker expression, cell phenotype, and spatial relationships in preserved tissue architecture. Classic examples include MICSSS, which performs iterative chromogenic IHC staining, scanning, and destaining on a single slide, and p16/Ki-67 dual staining, which uses chromogenic co-detection to identify cervical cells with combined cell-cycle deregulation and proliferation signals.

Fluorescence in situ hybridization detects specific DNA or RNA sequences inside fixed cells or tissue sections by hybridizing fluorescently labeled nucleic-acid probes to complementary target sequences, allowing the target’s copy number, chromosomal position, spatial distribution, or transcript abundance to be visualized microscopically. DNA-FISH detects genomic loci, chromosomal gains/losses, amplifications, deletions, and rearrangements, while RNA-FISH detects RNA molecules or transcript localization; in cancer cells, mouse tumors, neurons, organoids, macrophages, or drug-screening samples, the readout is fluorescent puncta, fusion/split signals, or localized RNA signal interpreted relative to validated controls.

Antibody-based immunofluorescence/immunocytochemistry detects the cellular or subcellular localization of a target antigen by binding a primary antibody to the target and detecting that antibody directly with a fluorophore-conjugated primary antibody or indirectly with a fluorophore-conjugated secondary antibody. Indirect immunofluorescence can amplify signal because multiple secondary antibodies can bind one primary antibody. The assay readout is fluorescence intensity and localization measured by fluorescence or confocal microscopy, and the result reflects antigen distribution only when the antibody has been validated for the target, sample type, fixation condition, and imaging workflow. Antibody specificity must not be assumed from catalog information alone, and appropriate validation or control experiments are required for serious interpretation.

The labeled nucleic acid probe is hybridized with the DNA or RNA on tissues and cells by using the complementary base sequence between the single strands of nucleic acid molecules. The detected DNA or RNA molecules in situ in cells can be displayed by autoradiography, fluorescence detection or enzyme color development.