immunohistochemistry and protein denaturation - (Feb/29/2008 )

Hi!!

I have a question concerning ihc and denatured protein...
I generated an Ab against a peptide and I tested it by Western blot (SDS-page so in denaturating conditions!). I detected the target protein!!!
I have a problem when I want to localize the protein onto tissue sections...I don't detect the protein!!!!!
My hypothesis is that is due to the conformation of the protein?... Is it the good hypothesis???

Is it a good idea to denature the sections (there were unmasked too)?
I know that I coud use Tris-HCl pH10 or urea... Is somebody try this??
Do you have an other idea to help me????

Thanks for all!!!

-Auror37-

QUOTE (Auror37 @ Feb 29 2008, 09:16 AM)

My hypothesis is that is due to the conformation of the protein?... Is it the good hypothesis???

Is it a good idea to denature the sections (there were unmasked too)?
I know that I coud use Tris-HCl pH10 or urea... Is somebody try this??
Do you have an other idea to help me????

Thanks for all!!!

What kind of sections are you using? What is your protocol? Also make sure you don't have azide in your antibody sample as this interefers with IHC.

-Clare-

QUOTE (Clare @ Feb 29 2008, 12:40 PM)

QUOTE (Auror37 @ Feb 29 2008, 09:16 AM)

What kind of sections are you using? What is your protocol? Also make sure you don't have azide in your antibody sample as this interefers with IHC.

I use paraffin sections (fixation with PAF 4% or Bouin) because histology is better than with cryostat sections! My purified Ab contains no azide.
My protocol is:
- deparaffin and rehydratation of sections
- unmasking on microwaves with citrate solution (vector)
- blocking endogen peroxidase with H2O2 0,3% in PBS
- permeabilisation with triton 0,1% in PBS
- blocking non specific with serum 5%
- primary antibody O/N at 4°C

-Auror37-

[/quote]

I use paraffin sections (fixation with PAF 4% or Bouin) because histology is better than with cryostat sections! My purified Ab contains no azide.
My protocol is:
- deparaffin and rehydratation of sections
- unmasking on microwaves with citrate solution (vector)
- blocking endogen peroxidase with H2O2 0,3% in PBS
- permeabilisation with triton 0,1% in PBS
- blocking non specific with serum 5%
- primary antibody O/N at 4°C
[/quote]

Is your protein localised in the nucleus? If it's not, you shouldn't need the citrate step. But if it is, how long are you unmasking for? I had to try different times when I was staining with an antibody I made (for a transcription factor). Have you stained sections with other antibodies successfully? Also, have you compared different fixation methods? Some antibodies prefer formalin to PFA. And you shouldn't need to do an overnight step for the antibody - 1 hour at RT should be fine - and saves heaps of time

-Clare-