Optimize HIER Antigen Retrieval for IHC

IHC Protocol for Optimizing HIER

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.
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:

Time	Antigen Retrieval Solution pH
	Acidic	Neutral	Basic
1 minute	Slide # 1	Slide # 2	Slide # 3
5 minutes	Slide # 4	Slide # 5	Slide # 6
15 minutes	Slide # 7	Slide # 8	Slide # 9

A similar testing method could be used to optimize the temperature. For antibody promotions and available IHC reagents, check out Boster's featured items below! For more IHC technical resources, just click this button:

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