[Histonet] pancreas pH or proteases inhibit LacZ enzyme activity?

[Collette, Nicole M.]

Hello, Histonet experts,

I have a puzzling bit of data that I am trying to resolve. Our group and another group have performed LacZ stain on mouse pancreas to identify cells expressing a LacZ reporter (the other group has published this expression pattern). I am having trouble with the primary antibody for the protein that LacZ is supposed to report for, so as a backup I tried an antibody for LacZ, which has worked very well for us in other tissues in the past, and has always matched the pattern we see with LacZ stains. With LacZ antibody, I get very robust stain in the islets, (as well as light stain in the surrounding tissue consistent with the LacZ stain pattern) and I see a qualitative dose response to diabetes induction, which makes me think the antibody stain is real. However, we and another group have not seen any LacZ activity in the islets, only in the surrounding tissue. Could the LacZ enzymatic activity be disrupted ex vivo by the pH of the islets themselves, or protease activity in the islets, so we can pick it up by immunostain but not LacZ enzymatic stain? Any other expert opinions as to what's going on? The insulin and glucagon antibody stains are working like champs, beautiful specific signal with very low background. I have been doing LacZ stains on various parts and pieces for the better part of 6 years, this is an unusual case where the confirmation controls didn't match the LacZ stain. And to clarify, I am doing the LacZ (and other) immunostains on pancreas sections that were NOT stained enzymatically for LacZ activity, but are the same genotype. I use Rene's recommended IM embedding protocol for paraffin. On the LacZ side, I stain whole-mount tissue prior to embedding and sectioning, with attention to fixation times and pH of paramount importance.

Thanks in advance for your help!
Sincerely,
Nicole Collette, Ph.D.
Lawrence Livermore National Lab
collette2 <@t> llnl.gov<mailto:collette2 <@t> llnl.gov>