[Histonet] Re: background

[Johnson, Teri]

Pablo writes:

>>Dear Histonetters,

I am doing OMP immunohistochemistry in paraffin-embedded sections of
sheep olfactory mucosa.

In my positive control -mouse olfactory tissue- there is not any
background, but in he sheep sections I get lot of unspecific labelling
(vessels, connective tissue, respiratory mucosae).

Being my primary antibody raised in goat I guess that there is an
undesired cross reaction with the sheep tissue.

Could I improve my blocking? I do half an hour in 2% BSA and 5% Horse
normal serum.

Should I use Goat normal serum?

Thanks in advance for your input.

Pablo Sanchez-Quinteiro
Lugo (Spain)<<

Luckily for you, Liz Chlipala covered this very topic in her absolutely
wonderful NSH teleconference today. All you attendees out there, look at
slide #48 and the answer is right there under her Antibody #2 example.

One possibility for this is that your anti-goat secondary antibody shows
minimal cross-reactivity with mouse, but considerable cross-reactivity
with sheep. Check your antibody spec sheet and see if it's listed there.
If not, call the company and ask them.

Adding normal goat serum to a pre-primary antibody blocking step will
most likely not have any affect if the problem is your secondary
antibody. And you definitely want to stay far away from goat serum any
time you are applying an anti-goat antibody to your tissues. You will
get high background staining all over if you do that.

Two possible fixes:
Change to an anti-goat antibody that has been cross-adsorbed for sheep
(or multiple species including sheep) and see if that fixes it.
-or-
Add 5-10% normal SHEEP serum to the diluent you use to make up your
anti-goat antibody, and incubate your dilution in the tube prior to
adding it to your sheep tissue.

Thanks Liz!


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