Protocol for Optimizing HIER Antigen Retrieval Conditions

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July 14, 2017
Dr. Booster
IHC Protocol for Optimizing HIER

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:
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

Antigen Retrieval Improves Detection of CD3E. IHC images show the detection of CD3E in paraffin-embedded human tonsil sections following incubation of tissue for 5 and 15 minutes at 95°C in the specified antigen retrieval solution. Compared to no HIER treatment, CD3E detection was enhanced following incubation in EDTA (pH 8.0), PBS (pH 7.2-7.6) and citrate (Ph 6.0) antigen retrieval solution. CD3E was detected using anti-human/mouse/rat CD3E polyclonal antibody (PB9093).
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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Citrate Buffer Powder 2L/Pack, For Heat Antigen Retrieval Antigen Retrieval Buffer (Enzymatic Digestion) For IHC Phosphate Buffered Saline (PBS) Powder
Citrate Buffer Powder 2L/Pack, For Heat Antigen Retrieval AR0024 Antigen Retrieval Buffer (Enzymatic Digestion) For IHC AR0022 Phosphate Buffered Saline (PBS) Powder AR0030
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