For my IHC protocol, I am staining for beta-catenin in ovarian sections during antigen retrieval. My samples are heated in citrate buffer for 45 mins.
I have also read that signs to show that your experiment is working fine is to notice the milky color of the citrate buffer after heating.
Why does the solution turn milky, and if it does not turn milky, does that mean your results would not appear?
Hi Mariam, Is this protocol for beta-catenin IHC taken from from the specification sheet which came with the antibody, or from the literature or are you following your general lab recommendation for HIER?
I perform antigen retrieval and i heat my samples in pressured vessel with citrate buffer for 4 mins and not more than that. I never encountered any problems with my results. I use brain sections. Follow abcam protocol.
I have done IHC on cervical cancer tissue as well as mice lungs. I have never faced such error.
There is nothing like milky solution is symbol for your experiment to be working fine,
Try to troubleshoot this buffer preparation, please check the prep. protocol according your vendor.
Check the pH of the buffer.
Try only heating the buffer, just to confirm that nothing else is interfering with the buffer.
I don't know your protocol, if you are following the heat & cooling cycles for antigen retrieval. If not then do try that.
One more thing, I have observed is that when the buffer is heated, is gets a bit cloudy but as soon as it cools down. It should turn back normal.
Hope this points help you to solve your query!!! Best of Luck
It is most likely the pH of the buffer. For a citrate buffer, you will want it to be around 6.0. Any higher and it will most likely disassociate sections of your fixation from the slide.
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