Sample Preparation: Tissue samples are collected and fixed using formalin or another suitable fixative to preserve tissue architecture and antigenicity. Sectioning: The fixed tissues are embedded in paraffin, and thin sections are cut using a microtome. Deparaffinization and Rehydration: Sections are deparaffinized and rehydrated through a series of xylene and alcohol washes. Antigen Retrieval: Techniques such as heat-induced epitope retrieval (HIER) are used to unmask antigens that may have been altered during fixation. Blocking: Non-specific binding sites are blocked using a blocking solution to prevent background staining. Primary Antibody Incubation: Sections are incubated with a primary antibody that specifically binds to the target antigen. Secondary Antibody Incubation: A secondary antibody, conjugated to an enzyme or fluorophore, is applied to bind the primary antibody. Detection: The enzyme or fluorophore is detected using chromogenic substrates or fluorescence microscopy, respectively. Counterstaining and Mounting: Sections are counterstained (e.g., with hematoxylin) and mounted for microscopic examination.
