JoVE Encyclopedia of Experiments
Cancer Research
0 views • 4:52 min • April 30th, 2023
- Begin by placing a thin tissue section in an ammonia solution to allow gentle antigen retrieval. Transfer the section to a polylysine-coated slide. Negatively charged cells bind to the positively charged polylysine. Next, add hydrogen peroxide to block the endogenous peroxidase activity. Wash the slide in Tris-Buffered Saline, TBS.
TBS promotes access of the antibodies to antigens by breaking the protein crosslinks. Wash the section with a block solution to reduce the non-specific antibody binding. Add primary antibodies to the section and incubate overnight. The primary antibody binds to its specific antigen present on the cell surface. Wash the slide with TBS.
Add Biotin conjugated secondary antibodies that attach to the Fc region of the primary antibody. Wash the section with streptavidin-biotin peroxidase enzyme. Next, add chromogenic substrate of peroxidase to the tissue. Peroxidase converts chromogen to a colored insoluble product.
Counterstain the tissue with hematoxylin solution. Hematoxylin binds to the acidic components of cells and turns them blue. Observe the tissue under a light microscope. The antigens are detected as red while the cells appear blue due to hematoxyl
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