Hello,
I'm having some trouble with visualizing my IF in spleen tissue. I first found extremely high background and could not differentiate fluorescent cells and background. I then realized my stained tissue looks no different than tissue that was imaged prior to any staining or fixation. Any advice would be greatly appreciated!
More detail on tissue origin (not sure what's relevant...) 15um sections from rats perfused with 4% paraformaldehyde. Tissue removed and stored in sucrose after 24 hr in para. Tissue was frozen in OCT prior to cryosectioning and slide mounting. I've tried a few recommendations with no success (glycine, ammonium chloride and trying overnight fixation).
Thanks in advance!
Brady! Is the auto-fluorescence laser/filter cube-specific? If so, you may want to switch secondary antibodies and avoid extensive trial-and-error problem solving.
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