[Histonet] HIER - best retrieval with minimum tissue damage?

From:"Nicola Cragg"


I'm a big fan of microwaving in citrate buffer as a method of heat inducing epitope / antigen retrieval.  However, I do run into problems when microwaving skin.  I've done a lot of IHC on skin and have found the microwaving is great when staining antigens in the epidermis, but I do have problems if the antigens I want to look at are in the dermis and require microwaving / heat for retrieval.  The tissue is often damaged and the morphology of the dermis is disrupted, so when I do see evidence of specific staining it's difficult to be confident in it.  

Obviously, when optimising antibodies, I simultaneously attempt it without antigen retrieval or via other methods such as proteolytic digestion, but some antigens in the dermis appear to require heat.  I have tried steaming, as I've read this is a gentler method, but I found it to be more damaging than microwaving.  I have had some experience with using a pressure cooker and a waterbath for HIER but have not yet tried it specifically for skin.  I use APES coated slides.  Does anybody have any suggestions?

Kind regards,

Nicola Cragg
Histology & IHC
EpiStem Ltd
Manchester, UK
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