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:
- Start with a neutral antigen retrieval buffer such as PBS (pH 7.2-7.6).
- 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)
- 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.
- 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: