[Histonet] Autofluorescence on Retina Tissue

[Allyse Mazzarelli]

Hi all!

Question for all you that may be more immuno-experienced than I:

I've consistently run immunofluorescence on pig retina and I seem to have a
severe case of autofluorescence/background. I've played around with primary
and secondary antibody ratios, but that doesn't seem to help my case. The
primary antibodies I use are goat-anti-FLT.1, and mouse-anti-rhodopsin. The
secondary antibodies I use are AlexaFluor donkey-anti-goat488 &
rabbit-anti-mouse555 (For my experiments, each slide contained only one
primary antibody, and it's corresponding secondary. For imaging purposes, I
did not double-label on these slides. E.g. FLT.1 was labeled with the 488
secondary, and rhodopsin was labeled with the 555 secondary).I re-hydrate,
conduct antigen retrieval, and block as per normal IHC protocol. However,
when imaging, I noticed that both slides, labeled with either rhodopsin or
FLT.1 seem to "bleed" through to the next filter. For example,
mouse-anti-rhodopsin labeled with the 555 secondary works beautifully at a
1:600 ratio. However, when I switch to the FITC filter on my scope, all the
retinal tissue appears green on the slides, even though it has ONLY the 555
secondary and NO 488. I've noticed this for the FLT.1 antibody as well
(i.e. switch to red filter and tissue fluoresces even though no slide saw
the 555 secondary antibody).

As I mentioned, I decreased the ratios of all antibodies, but that still
doesn't eliminate the problem.

If anyone has any ideas as to how I go about fixing this, please let me
know. I've only been in the field for a very short period of time, so if I
missed something in my description, don't hesitate to ask! Thanks for
whatever help you can direct my way!