IHC-P paraffin staining protocol - detailed guide

A guideline procedure and tips for staining of paraffin embedded sections, including antigen retrieval, chromogenic detection and fluorescent detection

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Immunohistochemistry (or IHC) is a method for demonstrating the presence and location of proteins in tissue sections. Though less sensitive quantitatively than immunoassays such as western blotting or ELISA, it enables the observation of processes in the context of intact tissue. This is especially useful for assessing the progression and treatment of diseases such as cancer. In general, the information gained from IHC combined with microscopy literally provides a “big picture” that can help make sense of data obtained using other methods.

Immunohistochemical staining is accomplished with antibodies that recognize the target protein. Since antibodies are highly specific, the antibody will bind only to the protein of interest in the tissue section. The antibody-antigen interaction is then visualized using either chromogenic detection, in which an enzyme conjugated to the antibody cleaves a substrate to produce a colored precipitate at the location of the protein, or fluorescent detection, in which a fluorophore is conjugated to the antibody and can be visualized using fluorescence microscopy.

IHC-P refers to the staining of tissues that have been fixed (usually in neutral buffered formalin) and then embedded in paraffin before being sectioned. The basic steps of the IHC-P protocol are as follows:

  1. Fixing and embedding the tissue 
  2. Cutting and mounting the section 
  3. Deparaffinizing and rehydrating the section 
  4. Antigen retrieval 
  5. Immunohistochemical staining 
  6. Counterstaining (if desired)
  7. Dehydrating and stabilizing with mounting medium
  8. Viewing the staining under the microscope

Contents

A. Optimizing a new antibody for IHC-P

  1. Antigen retrieval 
  2. Primary antibody concentration
  3. Detection

B. Fixation

C. Deparaffinization

D. Antigen retrieval 

  1. Buffer solutions for heat-induced epitope retrieval
  2. Heat-induced epitope retrieval methods
  3. Enzymatic antigen retrieval

E. Immunohistochemical staining 

  1. General guidelines
  2. Protocol
  3. Controls
  4. Signal amplification

F. Resources

G. Additional buffer recipes