c-FOS problems-troubleshooting failure?

[badger4science]

Hello all!
We have used the same company now for years regarding our source of rabbit anti C-FOS. As of the past few months we have tried several lots and staining is nil.  1 to 10,000 dilution.  No signal, some background with dendritic type detection, horrific.

It's a standard protocol: 4% fixed tissue, 20% sucrose cyroprotectant, 30 micrometer slices. .01M PBS in free floating wells,  MeOH/H2O2 endogenous peroxidase quench.  Wash, incubate in 4C for 48 hours, wash again, 1:200 Vector goat anti rab with NGS, wash, ABC incubation, DAB detection.

Where are we failing? It truly appears to be the antibody but I can't fathom why this would be. This company is huge.

The ABC and DAB react fine- other ICC runs with the goat anti-rab have worked swell.

[CanuckPhD]

Have you tried different dilutions of the antibody? Perhaps the antibody concentration is different from your previous batches and will have to be used at a lower concentration. Has the company explained any changes in the antibody?

Is your positive control working? That is are known positive tissues giving no staining?

Also you may want to try a Western blot to see if your antibody is detecting the protein. Or try some ICC on cell lines known to express the protein.

If all elese fails try a different supplier.

Good luck

[sleepy9]

I experienced similar situation like yours a year ago. It turned out to be the primary antibody had some problem; it wasted me several months worth of work. The c-Fos antibodies varied from lot to lot. I changed the source to a different vendor (Calbiochem) and it worked without problem. Contact your current vendor and they might send you a replacement antibody of different lot at no cost. If not, go for a different source.

[Emulated Reality]

Don't know if you are still having problems with the protocol, but in case you or anyone else is still curious...

I run a 1:3000 concentration on the primary overnight (around 21 hours), 1:400 on the Biotinylated secondary for an hour. I have also removed the H2O2 quenching step as I found it to be unnecessary and instead I run double rinsing steps between each incubation, ABC step, or DAB step. Low background and very clear specific staining. This method is also effective for a double stain using IF, for instance, simultaneously.

I can be more specific if anyone still needs help. I would guess that your primary concentration is too low or the quenching step is giving you problems.