Protocol Online logo
Top : New Forum Archives (2009-): : Immunology

colon immunohistochemistry - protol for immunohistochemistry on colon (Aug/28/2009 )

I'm trying to see a nuclear protein on paraffin embedded mouse colon tissue, but don't get any good result. I Know that this is not due to the antibodies because they work very well in other tissues so I'm wondering if colon has some special requirements..maybe different antigen retrieval steps...
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 4C o/n
Washes with PBS + 0,1 % Triton
Fluorescent secondary antibody diluted in blocking solution 2h RT
Washes
Mounting on slides

This protocol workes with many other tissues I tried...I don't understand which is the problem with this one....

Thank you!

Bye

-Velella-

What is the actual problem? If it is high background, it may be because you are using the wrong secondary antibody or a primary that is raised in a mouse. This could be the case if the other tissues that the antibody works fine on are human... and you are now switching to mouse.

-bob1-

The problem is high background, all the tissue gets the same staining and I can't see anything specific, I've always been using this combination of primary and secondary antibodies only on mouse tissues and they always worked well, the primary antibody is a rabbit anti mouse and the secondary is a donkey anti rabbit, so I don't see where the problen is....the colon is the first tissue which gives me problems, this is why I was wondering if it has something different from other tissues....

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.

-Velella-

Yeah, your antibodies should work fine with that combination. Are your tissues normally paraffin embedded? If not, this is often a problem too, the fixatives often don't wash out and cause non-specific fluorescence. If you know what they were fixed in it will help, glutaraldehyde is pretty much impossible to remove, but formalin is OK. Try washing in 0.1 M glycine over several days, changing the glycine wash every few hours (overnight will be fine, so you can get some sleep). I have used this before on sectioned formalin fixed tissue to reduce background, and my boss tells me this is the best bet for glutaraldehyde, but most antibodies won't work well on glut fixed tissue.

-bob1-

I usually fix tissues in Paraformaldehyde 4% in PBS for 24 hours, then I keep the tissue for 24 hrs in PB at 4C, then 24hrs EtOH70%, 24hrs EtOH 80%, then 3 hrs EtOH 95%, 3hrs EtOh 100%, 3hrs Xilene, 3 hrs Paraffin and then paraffin embedding.

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?

-Velella-

Sorry for slow reply, I was at a conference...

I do the washes on the sections on slides, there isn't enough permeability in a chunk of tissue for it to work well.

-bob1-