Hello. I am looking for something to stain brain cryosection tissue that is visible in normal light microscopy, but does not yield a significant signal with fluorescence microscopy (in range 405 to 647 nm). Gentle treatment of tissue is also preferred.
The reason I need this is to have a counterstain that our Zeiss Slide Scanner automatic microscope can use to automatically detect tissue focus (focus map generation), but I cant use e.g. DAPI because I'm using all 4 fluorescent channels for antibody staining.
Hi Anders, Hematoxylin (as far as I know) stains nuclei, but is not fluorescent.
Anders, Sudan Black may work for this purpose but if your immunolabeling is for antigens in white matter, then Sudan Black may not be optimal depending on your antigens of interest. Sudan Black is a myelin stain and is quick and easy to apply to frozen sections right after you finish your immunolabeling. It is often used to help suppress tissue autofluorescence in immunofluorescence experiments. The procedure is very simple (I've attached the protocol that we use). You can overstain the sections with Sudan black so you need to be a little careful to make sure you don't start quenching your specific immunofluorescence signal.
Some people have reported that Sudan Black can fluoresce in the deep red channel, but we have not experienced that in our own work.
Good luck.
Dave Sherry
Hematoxylin will fluoresce red so it is not good (same true for Cresol violet. Probably the only option is to use an optical method (Nomarski, or Phase optics to see material that is unstained.
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