Can anyone give suggestions for reducing the autofluorescence/background fluorescence of whole eye cryosctions in immunohistochemistry. The area of interest is retinal photoreceptor layer.
Hi,
You can try blocking your tissues for a longer period of time (1 hour tends to work pretty well for our lab), using a lower concentration of secondary antibody, or lowering the exposure time once you go to image your tissues.
Hi, if you can avoid formaldehyde fixation, (using a different fixative), autofluorescence is significantly reduced.
Kind regards, Daniela
If you're using only fluorescent (non chromogenic) readouts, you could also look into techniques like BrainBLAQ or even some mild clearing, those always help cut down background from autofluorescence especially!
Since photoreceptor opsins have a rather intense autofluorescence in both red and green channels, one thing I would suggest you is to try using secondary antibodies with far red fluorophores.
Best luck!
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