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Troubleshooting guidesProtocol for Optimizing HIER Antigen Retrieval Conditions | |||||||||||||||||||
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:
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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). |
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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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