Hi,
Our lab started using tdTom mice for lineage tracing. Currently we are using 2 antibodies to detect tdTom one from Clontech and other from rockland. When we use tdTom antibody for immunostainings, it detects the protein in the mouse tissue, but when we have to do citrate buffer antigen retrieval, we loose the fluorescence or we get a very faint staining.
It is becoming difficult to find lineage traced neurons when we have to pair the tdTom with other markers if we need antigen retrieval.
Any suggestions regarding good antibodies would welcome!
Thank you
Raghu
Hi there,
Most likely your problem here is not the antibody since it works well in non ag retrieval conditions. Assuming that you are using heat-induced antigen retrieval (HIAR) with citrate buffer, it is well known that the antigenicity for some epitopes is lost during this process. In my experience, while HIAR is advantageous for membrane proteins (ion channels, neurotransmitter receptors,...) it clearly compromises the labeling of (some) cytoplasmic proteins.
This review is really helpful regarding IHC and Ag retrieval:
https://pubmed.ncbi.nlm.nih.gov/24129895/
I just have two suggestions:
1) Try to modify fixation conditions to get the fixed TdTom more resistant to the lost of Antigenicity induced by HIAR (Caveat: If you fix more strongly the tissue, you may also be facing higher antigenicity lost in your protein of interest)
2) Try a a different method for Ag retrieval, maybe proteolytic-induced Antigen retrieval with pepsin could help you to solve your issue.
Hope this helps,
J
