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:
|Time||Antigen Retrieval Solution pH|
|1 minute||Slide # 1||Slide # 2||Slide # 3|
|5 minute||Slide # 4||Slide # 5||Slide # 6|
|15 minute||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).[/caption]
A similar testing method could be used to optimize the temperature. For antibody promotions and available IHC reagents, check out Boster’s featured items!