What are the general steps involved in immunohistochemistry (IHC)?

The main steps involved in immunohistochemistry (IHC) include: 

• Tissue Preparation: The first step involves fixation, embedding, and sectioning of tissue samples. Fixation maintains cell and tissue morphology, increases the refractive index of tissue constituents and prevents autolysis and necrosis of excised tissues. Embedding helps preserve tissue morphology and supports the tissue while it is being cut into thin sections, which is essential for facilitating more detailed study.  
• Antigen Retrieval: Antigen retrieval helps to recover masked epitopes and facilitates an antibody’s access to the target protein within the tissue. 
• Permeabilization: Permeabilization is necessary to help antibodies access the cell’s interior to facilitate detection of target antigens, including cytoplasmic epitopes of transmembrane proteins and intracellular proteins. 
• Blocking: IHC procedures typically encompass one or more blocking steps, including protein blocking, biotin blocking, and endogenous enzyme blocking. Blocking helps to diminish background signal and prevent false positives. 
• Immunostaining: This may be done either via direct or indirect detection. The direct detection method involves directly conjugating the primary antibody to a label. The indirect detection method involves binding the primary antibody by a labeled secondary antibody that has been raised against the host species of the primary antibody. 
• Visualization: Antibody complexes are visualized using chromogenic or fluorescent substrates.