Hi All,

I've been using a primary antibody (anti-BMP4 from R&D Systems) for immunohistochemistry on mouse testis tissue. Everything was working fine, then all of a sudden, it stopped working. I've been using all the same reagents and conditions, and I havent changed my batch of antibody (same batch but different aliquots that I reprared myself when the antibody arrived). As far as I can see, nothing has changed in the protocol, yet I cant get it to work.

I was wondering whether this sort of thing is common? I dont have alot of experience with immuno work and would like to know it this is to be expected.


Thanks...

rmb

Hi,
have you been doing a double immunostaining? The second Ab, or enzyme might have failed. If you haven't checked it yet, try IHC with different primary Ab about which you are sure that is working. I am not an expert, so my advices may be useless
Good luck

Are you working with paraffin-embedded sections? If so, maybe you need to change your deparaffinization solution (xylene) or rehydration solutions (alcohols) or your antigen retrieval solution.

Frequent thawing of antibodies can degrade antibodies.
Also any solution used for the immunostaining could cause failure. Blocking solutions, fixatives, permebilisation, etc.

Best to get fresh aliquots of antibodies and prepare fresh solutions.

Good Luck !!!

I'm having the same problem as rmb and also don't have a ton of experience with immunostains.
I know it's my primary Ab that is failing, because other primary antibodies (using same reagents for blocking, permeabilisation etc.) are working. The secondary Ab that I have been using is working as well, tried it with a different primary Ab and got specific staining.
When I first started this project and started working with my Ab my boss suggested to add glycerol so we could save it at -20 degrees C, even though the Ab datasheet clearly states you should keep it undiluted at 4 degrees C. Since they have a lot of experience it seemed like the right thing, although I'm staining tissue sections and their experience is with cell cultured monolayers. I'm not sure if that makes a lot of difference in how good the Ab should be.
Could it be that the Ab is just not working anymore (maybe bec/ I ignored the data sheet)?

Thanks in advance.

Ddkb on Feb 18 2009, 07:03 PM said:

Hey mdfenko,

Thanks for your response. The antibody didn't freeze, but I was still wondering if adding the glycerol might have done something to it. Even though the first few times (about 6 months ago) it was working fine and at that point it also had glycerol in it.
And yes, I'm going to buy a new vial and store it at 4 degrees as they suggest...

Still wondering though why it's not working anymore.

Thanks,

as with most (all?) proteins, antibodies will denature (we call it "crap out") over time.

since it didn't freeze and worked for some time after the addition of glycerol, i would consider storing in glycerol at -20C in aliquots small enough that you can finish them within a few uses (if not single use). this will avoid changing conditions too many times (allowing it to warm then cool again over and over and ...).

on the other hand, we have stored antibodies at the conditions recommended by the supplier and thay have remained useful for quite some time.

we also store the antibodies that we make ourselves (not conjugated with enzyme) frozen (without glycerol), in aliquots, and they last for a long time.