gelatin - triton

From:Andres Garelli


I'm doing IHC on rat eye sections. And I have some questions

1) As I don't have much experience, I had to cut thick sections (8 um) to obtain good ones. I tried some stainings with the antibody (for a transcription factor, that is, with nuclear localization) with frustrating results.
But the antibody works well on cell cultures, so it wasn't the antibody. So tried with a pre-treatment with triton100X 0,1%, as I do with the cell cultures to permeate the cell membrane.I thought that perhaps as the sections were thick, the nuclear membrane was intact and the antibody could not cross it. But then was when the tissue detached from the glass!!!

2) As tissue detaches from glass slides, I decided to coat the slides with chrome gelatin. Would this be a good procedure for IHC, or would the gelatin produce background?
I think that silanized slides would be better, but I don't have access to that reagent.

3) I said I can't use APES, but in our lab we use poly ornithine for cell culture. As it has almost the same structure as poly lysine (their lateral chains differ in one C atom length and both have a primary amino group) I asume it should they should have almost the same pKa. And so, I thought that I could replace poly lysine with poly ornithine. Would this work?
Would I overcome the backgroung fluorescence of gelatin? (in case gelatin produces background).

Thank you very much.

Andres Garelli
Instituto de Investigaciones Bioquimicas de Bahia Blanca - INIBIBB
Camino La Carrindanga km 7
CC857 - B8000 FWB
Bahia Blanca
Tel: +54-291-4861201 (interno 136)

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