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.