I'm troubleshooting immunofluorescence staining for some antibodies. Some work fine with my protocol [H2AX, Ki67, 3-nitrotyrosine], some stained once and never again [8oxoG], and some won't stain at all [Cleaved Caspase-3, malondialdehyde].
I often see that you should preheat your antigen retrieval buffer before adding slides, but I use the 2100 Retriever pressure cooker which does not call for preheated buffer. Does preheated buffer work better? And why?
Also, does anyone have suggestions about what to try next? We've played with permeabilization time and buffers, antibody dilution, and both HIER and enzymatic antigen retrieval.
Thanks!!
Jennifer,
I have always preheated my buffer when working with a steamer. I do this simply so that the entire time the tissue is in the cooker, the temp is 90 degrees celsius. Otherwise, it may take a few minutes for the solution to reach the desired temperature in the steamer, resulting in less time overall retrieving antigens. If you are really concerned about this, you can simply increase the time of your antigen retrieval. I know some people preheat in a microwave for a minute before the steamer, although I have never tried that. I would suggest that this is probably not the problem you are having. Is your tissue fixed? If so, for how long? In what fixation solution? Do you do any cryoprotection? Without knowing these things it is hard to say for sure....but I will say this....9 times out of ten...these issues come down to tissue quality/overfixation when antibodies are inconsistent. I would suggest a 4 hour post-fix (definitely no longer than overnight)and then transfer over to a 30% buffered sucrose solution until cutting and staining. Any sliced tissue not used within 1-2 weeks should be placed into a long term cryoprotectant and stored at -20.
Renee
we preheat the buffer in microwave oven for a minute or so before cooker and it works fine. I have had one case which I forgot to preheat the buffer and it didnt work. it can happen but depending on the samples as well as antibody normally these issues raise when either there is problem with the tissue processing or the antibody itself. if your tissue sections are old, prep some new ones. sometimes the antibodies stop working, simply due to being and old batch. and some antibodies doesnt work at all. so you may even need to change the clone of the antibody or the purveyor. we have a Cleaved Caspase-3 from cell signaling which works brilliantly with FFPE sections following antigen retrial using a citrate-based buffer for 10-15 mins. I can share our protocol with you if you need.
It is HIER (heat induced epitope retrieval). you need to have your slides in HOT buffers, either citrate pH6 or EDTA pH8 for a prescribed time. I set my nice and stable water bath at 97.5C. When you add slides you will get a temperature drop. For IHC staining you need to be un-mithered, precise, organised, (verging on OCD). Are you using a KNOWN positive control tissue? Are you sure it is your "technique" or your "reagents" and not your samples? You will also need to do other controls as well. Perhaps it is your primary?
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