[Histonet] Re: Immunohistochem help!

[Johnson, Teri]

Graham, the short answer is: it depends (on whether the antigen you're
trying to demonstrate is recognized by the antibody after formalin
fixation). For most of our IHC in frozen section, we use formalin fixed
and sucrose cryoprotected frozen sections. We do use antigen retrieval
for some of the antibodies, notably BrdU and GFP, but at 60 degrees C
for 10 minutes. Some epitopes (proteins) become more stable with
fixation prior to immunostaining, while others undergo enough
conformational changes that the antibody does not recognize it. In many
cases polyclonal antibodies might be more "forgiving" and therefore
easier to detect the protein regardless of the pre-IHC processing.

The general protocol we use is basically the same as with paraffin
section with three notable differences:

1 - no deparaffinization step
2 - Antigen retrieval is done at a lower temp (always test with and
without - sometimes you will destroy the signal using AR)
3 - H2O2 quenching is done using 0.3% hydrogen peroxide in buffer or
water for 30 minutes @ RT.

All other procedural steps are the same.

Hope this helps!

Teri Johnson, HT(ASCP)QIHC
Managing Director Histology Facility
Stowers Institute for Medical Research
1000 E. 50th St.
Kansas City, MO 64110


Dear All-
 
I'm a student with a little bit of experience in immunohistochemistry. I
am familiar with using paraffin sections and fresh frozen sections,
however i've never stained any tissue that was fixed prior to freezing.
I was wondering if anyone has a general protocol for staining fixed
(with formalin) frozen tissue. Is it better to use fresh tissue for
frozens rather than using fixed frozen tissues? Thanks in advance!
 
Graham Brown
 
Texas Tech University Health Sciences Center


Teri Johnson, HT(ASCP)QIHC
Managing Director Histology Facility
Stowers Institute for Medical Research
1000 E. 50th St.
Kansas City, MO 64110