Is there such a case that certain antibodies have limited access to the intra-cellular domain of its receptor and this results in a negative staining?
I have been struggling trying to detect signals associated with the intra-cellular β-subunits of the insulin-like growth factor receptor (IGF-1R). Using exactly the same protocol however, I got very good results showing a positive staining of extracellular α-subunits of IGF-1R.
I have tried all the commercially available antibodies against IGF-1R beta-subunit which are applicable for immunohistochemistry, so I was wondering why this is the case.
Also, if my IHC results showed positive staining of α-subunits, can I conclude that the whole receptor is present, rather than saying only the α-subunits of the receptor are present?
It would help if you could share your staining protocol to rule out trivial oversights such as insufficient cell permeabilization ...
Dear Yinan,
As the target is a specific sequence present in the midst of the 1333 to 1366 AA at the c-terminus of IGF-IRβ subunit, localizing it could be a problem if the sequence is getting denaturated for some reason.
1- whatever the cell-line you are using do not detach the cells by trypsinization. Keep the flasks on ice for 30 mins and then tap vigorously by hitting the flask to a wooden surafe gently. Use the physically detached ones for IHC/ICC.
2- . Insufficient cell permeabilization could be an issue as mentioned by Lars.
3- If these dont work, you can validate IGF-IRβ by tyrosine kinase inhibitors, I guess.
Will keep you posted on this in 24 hrs if i get something new.
Best,
AMB
Hi all.
Thanks for the reply. I am actually doing IHC on formalin-fixed paraffin embedded mouse tissue sections, not cells.
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