IHC on Hormones of the Taste Bud - Need some direction (Feb/23/2010 )
I am currently attempting to perform IHC and IF on peptide hormones of taste cells in the circumvallate papillae (CVP). We have successfully located the peptide using IHC in its control (pancreas) using trypsin and no antigen retrieval but the same protocol does not work for the CVP We tried multiple dilutions for the antibody 1:50, 1:100, 1:150 and still no difference. We have just switched our fixation method to fresh frozen in hopes we will get a different result.
Similarly, we are trying to locate the receptor for this peptide in both its control and in the CVP. We have failed to do so in both occasions.
I was wondering if anyone has any information on a better protocol to use SPECIFICALLY for hormones of the taste cells. We are having trouble capturing the peptide in its granules in the cytoplasm of taste cells. It has been shown that there seems to be a robust peptide expression but low mRNA levels for these peptides in the taste cells. If anyone has any information on a better direction to shoot me in that would be great. If there is any other information you guys need don't hesitate to ask. Thank you for your time.
did you try neat antibody and longer incubation period? Try with and without trypsin and vary incubation period of the enzyme.
Well so far we have tried only overnight incubation. We will try extending it to 24, 48, and 72 with this new fresh frozen tissue and see how it goes. Thank you for your input.
sgt4boston on Feb 23 2010, 04:08 PM said:
Do you mean neat as in undiluted? IF so that is not practical for the type of anitbodies we are using.
Neat is undiluted. If you were not getting any signal you can try the maximum concentration you can afford to do with an extended incubation.
to confirm that all the other reagents are working properly is there another marker/ab system you can run in parallel on the same tissue?