I've previously done multi labelling immunofluorescence experiments using 405 conjugated secondary antibodies, which worked well. This was in non perfused mice, where the brain was sliced in oxygenated aCSF and then slices fixed in 4% PFA for 30 minutes.
I'm now working with 4% PFA perfuse fixed brains, which are postfixed overnight in 4% PFA before being incubated in 30% sucrose solution until they sink, before slicing on a vibrotome. The antibodies now show high non specific staining of vessels and cell bodies. I've tried including glycine in immunofluorescence protocols to reduce any PFA induced autofluorescence, to no effect. Additionally, I tried heat mediated antigen retrieval to 'de-fix' the tissue a bit to see if this could perhaps help, which it did not. My only thought has been that perhaps its lipofuscein in the tissue, as that supposedly accumulates with age and is autofluorescent, but the mice I'm using now are not significantly different in age from those I used previously when the 405's were working.
Any thoughts would be greatly appreciated! I have also considered a different conjugated secondary antibody, but with our current conditions and lasers available, 405 seems to be the only option.
Attached is a .pdf I use that has protocols, especially on pages 6 & 7, to eliminate fixative-derived non-specific staining and autofluorescence. Hope the protocols are helpful, best of luck in your experiments.
Thanks Sara, I think I solved my issue, which seemed to be PFA related. I treated my sections with ammonium chloride and glycine before the blocking stage (versus glycine in the blocking step) and this has revealed specific antibody signal in the 405 channel. I also tried CuSO4 in ammonium acetate buffer and a commercially available autofluorescence quencher from EMD millipore to combat lipofucin autofluorescence, and while this helped it didn't work as well as removing PFA autofluorescence
- Badges
- Science method