1. Tissue Preparation: The tissue is fixed and embedded, usually in paraffin. 2. Antigen Retrieval: Heat or enzymatic treatment is used to unmask epitopes. 3. Blocking: Non-specific binding sites are blocked to reduce background. 4. Primary Antibody Incubation: The tissue is incubated with primary antibodies specific to the target antigens. 5. Secondary Antibody Incubation: Fluorophore-conjugated secondary antibodies are applied. 6. Visualization: The stained tissue is visualized using a fluorescence microscope. 7. Image Analysis: Software is used to separate and quantify the different fluorescent signals.
