I have a piece of paraffin-fixed brain slide, which I hope to perform both immunofluorescent staining and cresyl violet staining together. Afterwards, I want to first image using fluorescent microscopy, then image under regular light microscopy. Is this CV-atop-IF staining idea possible? I have been trying to find literature that has done it before, but I couldn't find anything.
Perhaps this article might be of use:
Creasy, B. M., Hartmann, C. B., White, F. K. H., & McCoy, K. L. (2007). New assay using fluorogenic substrates and immunofluorescence staining to measure cysteine cathepsin activity in live cell subpopulations. Cytometry Part A: the journal of the International Society for Analytical Cytology, 71(2), 114-123.
You should be conscientious in picking an antibody that does not fluoresce at the same wavelength as the cresyl violet. Otherwise, it will be difficult to differentiate the staining. Starting with fluorescent and following with regular light (as you implied) is the most logical order. Of course, testing a piece of your sample, or a similar sample, is a good place to start to see if your particular antibody, the tissue itself, and the cresyl violet might have a unique interaction.
this is a good question. CV is very easy to do and requires few seconds and then a rinse or 2. Though we did a lot of work on CV and fluorescence, we never combined the 2 methods. What matters for your question is that fluorescent staining stays during the dehydration steps, needed for processing paraffin sections. Some may say never dehydrate Cy2, Cy3 (A488-A568-84) -stained sections but it work very well. Here is our 2012 paper where we did a lot of Cv and a lot of fluorescence (on PFA-fixed frozen section, which are more fragile), and then sections were processed as for paraffin (dehydrate, xylene-DPA mounting medium). It works very well. Article Long-term survival and differentiation of retinal neurons de...
,Article Long-Term, Stable Differentiation Of Human Embryonic Stem Ce...
,CV staining was done the same way. So, i see no problem except for potential fluorescence of CV (which i am not aware of; this is potentially a good point and you have to check). However, again, there is a method of taking CV or H&E images using fluorescent microscope rather than brightfield (you take B, G, R channels and then merge, somebody even published on this method). What you get is a nice RGB image, which we all like to see. So, as previous person said, do you fluorescent imaging (which requires de-waxing -and then staining with primary-secondary, and DAPI counterstain), then try adding a drop of CV on the slide, keep for 10-30 secs,rinse twice with PBS), then dehydrate the slides, pass through xylenes, add DPX, image and post the image for us. Both Fluorescence & CV will stay . Article Long-term survival and differentiation of retinal neurons de...
,Article Long-Term, Stable Differentiation Of Human Embryonic Stem Ce...
,For color image acquisition using fluorescent microscope read some good tips here, i think these are the papers i am referring to. Have an open mind, good question
https://carpenterlab.broadinstitute.org/blog/quantifying-microscopy-images-top-10-tips-for-image-acquisition
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