I need to stain tissue for octopamine and synapsin together and need to avoid wavelengths between 450-500 as my tissue gives too much autofluorescence in this range. I cannot go lower than 400 so I am pretty much out of options.
This is reasonably straitforward if you have access to a spectrophotometer. The relationship may then be quantified as a linearized matrix cross dependency. See my RG post " Modelling Multi colorant Subtractive Mixture..." or Roy Berns' paper
"A generic Approach to Color Modeling" in Color Research and Application Vol 22 Pages 318 - 325
Hi, with the right dichroic and filter set you absolutely can image those two together. 561 and 633 are right around the very commonly used red and far red channels.
Hi Margarita, you can decrease the autofluorescence by incubating your tissue samples with 1 mM NaBH4 in PBS for ~30-60 min at RT.
Autofluorescence in fixed tissues is mostly due to reaction of aldehyde fixatives (PFA, Glutaraldehyde, etc) with proteins to form fluorescent Schiff base products.
Even glucose (an aldose) forms fluorescent products with proteins. They are prevalent in diabetes and are called advanced glycolsylated end products (AGEs).
What these products have in common are fluorescent double bonded N and O adducts, which NaBH4 can reduce and render non-fluorescent.
Here is one example of NaBH4 reduction of autofluorescence:
Article Reduction of background autofluorescence in brain sections f...
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