I am trying to stain a membrane protein, but the staining failed for an unknown reason. After applying the secondary antibody (Alexa 647), I did the postfixation with %3 PFA + 0.05% glutaraldehyde. Can I stain the sample again now?
You certainly can try. However, the reasons why the staining failed should be figured out to give a better judgement here.
Make sure that there is no Alexa 647 back ground signal comparing to your control sample.
It would probably end up to be a long shot. You could think about antigen retrieval. In that case you would have to hope that your epitope of interest is heat stable and not masked by glutaraldehyde(GA). As GA will be more resilient to heat than the PFA. 0.05% GA is relatively low concentration so their are good chances that it will work.
However, you should keep in mind that it might be easier to run the experiment a second time. You should always keep reproducibility first. If you invest more time and chemicals in this particular sample, it should be really worth it. If you are not successful, you will learn nothing. Not only if you get an unexpected result you would have to repeat the whole process (including the failed double staining) to get to the bottom of this…
Good luck!
I will recommend to repeat from the scratch. and also thin if you altered any step in first attempt
Hi Qilin Yu . You can try quenching and restaining the sample as described here:
Multi-Phenotypic subtyping of circulating tumor cells using sequential fluorescent quenching and restaining | Scientific Reports (nature.com)
https://www.nature.com/articles/srep33488
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