Inconsistent IHC - Looking for help (Mar/05/2010 )

We recently got a homemade published antibody aliquot from another lab. I titrated the antibody to work at 1:2000 in frozen sections. The next day, I attempted IHC with this antibody in frozen sections at 1:2000 and my background was huge. Thinking that maybe it was the tissue, I titrated the anitbody in the new frozen tissue, and I found that 1:2000 and 1:4000 work. So the next day I did another experiment on this tissue, with both concentrations (1:2000 and 1:4000) just in case. Both concentrations worked well. Still had some background, but was acceptable. The next day, same tissue, used 1:4000 dilution and then again get super high background.

Here's my protocol, which the lab has used consistently for other antibodies in frozen tissue for years:
1. Dry slides
2. Fix 4% defrosted PFA at RT for 5 min.
3. PBS-T 3x5'
4. Block 1-2hr
5. Primary antibody O/N RT
6. PBS 3x5'
7. host-biotin at 1:200 for 1hr at RT
8. PBS 3x5'
9. biotin-SA at 1:100 for 30m at RT
10. PBS 3x5'
11. TSA amplification for 10m at RT
12. PBS 3x5'

Any ideas where this background is coming from? Someone in lab suggested that homemade antibodies are not homogenous, so maybe that's where the problem is, but that sounds somewhat dubious to me.

Thanks.

Did you re-use the antibody solutions when you did your titration and then fresh when you did the 1:4000?

If so, homemade antibodies are often a bit dirty, using them once or twice on non-important slides or blots can remove the non-specific binding/background. The same applies for secondaries.

bob1 on Mar 7 2010, 06:34 PM said:

Antibodies will also sometimes clump and precipitate themselves; some manufacturers recommend vortexing them briefly to resuspend the ppt.

If you were provided with a highly purified mab it is possible to precipitate over time.
Vortexing may not get the ab back into solution if you see a precipitate.

You mentioned "homogenous"...do you mean not purified (IgG fraction) or not affinity purified (purified on ag-column)? This part of your test did not change so whatever background you saw initially would still be there.

Is the tissue, host-biotin and biotin-sa working properly (in other tests?)

A better method to express use of ab is by concentration and not dilution (ie ug/ml)

I have the answer!!!!

Turns out that our lab tech who makes the block overheated it, causing it to inactivate most of the proteins. We aliquot and freeze our block, and each aliquot was ok or bad, depending on which part of the batch she poured it from. Making new block completely saved the IHC.

What poor timing and a waste of a month! But I'm glad it's back to working.

-UMich Grad Student

so it was the tissue component of your test.

glad you discovered your problem