Posted 28 August 2009 - 06:23 AM
Is there anybody who does IHC (fluorescence) on colon paraffin embedded tissues who can help me?
briefly my protocol is the following:
paraffin embedded colon slices (cut 10 um)
deparaffinization with xilene, then rehydration with ethanol 100%, 95%, 80%, 70%, water
Antigen retrieval with citrate buffer 10 mM pH 6 15' microwave max potency
water room temperature 30'
PBS+0,05% Triton (permeabilization)
blocking solution (I usually use the powder provided with the Perkin Elmer TSA Biotin kit) 1 h
Primary antibody diluted in blocking solution 4°C o/n
Washes with PBS + 0,1 % Triton
Fluorescent secondary antibody diluted in blocking solution 2h RT
Mounting on slides
This protocol workes with many other tissues I tried...I don't understand which is the problem with this one....
Posted 30 August 2009 - 04:23 PM
Posted 31 August 2009 - 03:14 AM
I verified the quality of my colon slices with an hematoxylin-eosin staining, and it was good. Then I tried a staining for beta actin on colon slices, and it worked a little better than the antigen I'm trying to see now, but I had always high background, so I thought that the problem could be "colon-specific" and only when you need an interaction antigen-antibody.
Posted 31 August 2009 - 02:11 PM
Posted 01 September 2009 - 06:50 AM
I will try with the glycine washing....I see that you write "sectioned" so did you perform the washes on the tissue slices attached on the slides or on the tissue before cutting it?
Posted 06 September 2009 - 05:35 PM
I do the washes on the sections on slides, there isn't enough permeability in a chunk of tissue for it to work well.