Tissue Preparation: The tissue sample is fixed, usually with formalin, and then embedded in paraffin. This helps preserve the cellular architecture. Sectioning: Thin sections of the tissue are cut, typically around 4-5 micrometers thick, and placed on glass slides. Deparaffinization: The paraffin is removed from the tissue sections using xylene or a similar solvent, followed by rehydration through a series of decreasing alcohol concentrations. Antigen Retrieval: To unmask the PCNA epitopes, the tissue sections are treated with heat or enzymatic methods. Blocking: Non-specific binding sites are blocked to prevent non-specific staining. Primary Antibody Incubation: The tissue sections are incubated with a primary antibody specific for PCNA. Secondary Antibody Incubation: A secondary antibody, often conjugated to a detection molecule like an enzyme or fluorophore, is applied. Detection: Depending on the secondary antibody, a chromogenic substrate or fluorescent dye is used to visualize the PCNA staining.
