Protocol for Optimizing HIER Antigen Retrieval Conditions

Greetings Earthling,

If you are performing IHC experiments, you may need to optimize your antigen retrieval methods. Techniques generally fall into two main categories: protease-induced epitope retrieval (PIER) and heat-induced epitope retrieval (HIER). In general, HIER has a much higher success rate and is recommended over PIER methods.

The protocol must be optimized for each sample tissue, fixation method, and antigen. HIER is especially time-, temperature-, buffer-, and pH-sensitive, and the best condition must be determined empirically. Here is a step-wise protocol that may be helpful:

1. Start with a neutral antigen retrieval buffer such as PBS (pH 7.2-7.6).
2. In order to exclude artifacts caused by the HIER process, results should always be compared to a control sample for which no HIER was performed.

   
   In this image, CD3E was detected using Boster’s anti-human/mouse/rat CD3E polyclonal antibody PB9093 (no HIER treatment)

3. If the neutral staining solution did not yield a good staining, alkaline or acidic antigen retrieval buffers should be tested. If you need an acidic antigen retrieval buffer, check out Boster’s sodium citrate buffer AR0024 below.
4. Ideally try various pH, temperature and time combinations. The table below depicts a typical experimental set-up for testing optimum HIER incubation time and pH: