I want to analyse formalin fixed and paraffin embedded tissue sections of human cardiomyocytes with immunofluorescence. Since formalin fixation disturbs protein antigens I need to retrieve them before staining with specific antibodies. Does anyone has experience which conditions fit best for cardiomyocytes (heat?, temperature? duration?) The protocol should ideally be easy to carry out - without the help of any special devices.
Thanks in advance,
I have done antigen retrieval for heart sections (paraffin).
I used Vector lab antigen retrieval solution and used the protocol what they have in data sheet. Never have problem. I Used 4 micron thick section on plus slides.
Need rice cooker and microwave.
Good luck
0.3% protinaseK at degree c in PBS(1M pH7.4) for 20-30 minutes serves permeation and antigen retrieval both. or alternatively you can go for heat antigen retrieval method i. e. slides dipped in citrate buffer pH 6 in pressure cooker for 30 minutes. both are validated and commonly used methods. go for it you will defiantly get success.
best of luck
I Have been using enzyme digestions for few of the epitopes and had fair amount of success. Keep in mind it is always a trial and error method. Pepsin digest all solution from InVitrogen (or you can make your own cocktail of trypsin and pepsin) 8 mints at 37 deg has worked for us. good luck.
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