Antigen Retrieval Protocols

Antigen retrieval refers to any technique in which the masking of an epitope is reversed and epitope-antibody binding is restored. Why is antigen retrieval necessary in immunohistochemistry (IHC)? The impaired ability of antibodies to access epitope in fixed tissue can impact IHC staining. Protein fixation creates crosslinks that mask tissue antigen and limit antibody-epitope binding.

Protease-induced antigen retrieval (PIER)

Antigen retrieval buffer for protease-induced antigen retrieval (epitope retrieval):

       2 mg/ml pepsin in Tris-HCl pH 2.0
      Or 0.5 mg/ml trypsin in Tris-HCl pH 8.0

Protease-induced antigen retrieval protocol:

1. Pre-warm the buffer to 37°C;
2. Keep the sections in retrieval buffer at 37°C for 10-20 minutes.
3. Suitable for antigens that are sheltered by fixative such as:
4. Collagen, GFAP, Complement, Cytokeratin, C-erB-2, LCA, LN, etc.

Heat-induced antigen retrieval (HIER)

Antigen retrieval buffer for heat-induced antigen retrieval (epitope retrieval):

10 mM Sodium Citrate pH 6.0

Heat-induced antigen retrieval protocol:

1. Microwave the antigen retrieval buffer until boiling and power it off, put in the sections;
2. Repeat step 1 for one or two more times for every 5-10 minutes;
3. Cool down at room temperature and wash with dH2O.

Preferred antigens:

Bcl-2, Bax, c-myc, E-cadherin, ChromograninA, Heatshock protein, HPV, P-glycoprotein, PKC, PCNA, etc.