Hi guys! Currently I am working with HIER on tumor tissue. I used 95dC 20m for HIER but the tissue kept falling off / folding.. I am suspecting that this is because of high temperature. Is there a protocol (60dC?) of lower temperature in dealing with HIER using Citrate buffer that effectively prevented tissue loss? Thanks.
This is normally not a major issue. You should not experience this issue with FFPE sections if you use Superfrost slides or poly-L-lysine coated slides. It is also important to preprocess the slides, i.e to bake the sections for 60C for 1 hour and dewax before HIER
Hello Haiyao Ding
For immunohistochemistry, we use Positive Charge Slides.
We do the antigen retrieval (after deparaffinization and rehydration with PBS) using a microwave as follow:
- Put the slides in 0.01 M NaCitrate pH 6.0 preheated for 5 min. Cover with plastic film, and microwave the sections at high power for 3 min at 900 W. Leave in oven for 20 min allowing the sections to cool.
Good luck!
Better to use decloaker (pressure cooker) than MW for the HIER. What tissue are you dealing with?
Hi Kristian E Moller, we are dealing with colon tissue and we used a water bath & polysine slides. However some big samples still peel off. Does baking longer (ie. overnight) of slides help? And if it does, what was the principle behind baking it?
Try 60C overnite, but do not exceed that temperature. That may resolve your issue
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