Achieving optimal binding involves several steps and considerations:
Fixation: Use appropriate fixation methods to preserve tissue morphology and antigenicity. Common fixatives include formalin and paraformaldehyde. Antigen Retrieval: For immunohistochemistry, antigen retrieval techniques such as heat-induced or enzyme-induced retrieval can enhance antibody binding. Blocking: Use blocking agents to prevent non-specific binding of antibodies. Common blocking agents include serum, BSA, and casein. Primary Antibody Application: Apply the primary antibody at optimal concentration and incubation time, following manufacturer recommendations. Secondary Antibody Application: Use a compatible secondary antibody conjugated with a detection enzyme or fluorophore for visualization. Counterstaining: Apply counterstains like hematoxylin to provide contrast and enhance visualization of cellular structures.
