I am setting up a differentiation assay from human adipose-derived stromal cells into different cell types. One culture grew on a (gelatin+laminin)-coated plastic support but not on the glass slide. Can I yet do immunofluorescence with my marker of interest or will the plastic give too much autofluo? Maybe red fluo (instead of green) would work?
We do that all the time. There is some auto-fluorescence but not problematic in our hands.
I have performed it many times on gelatin-polylisine, both on glass and on plastic, with no problem at all.
It works in tissue culture plates (12 wells, 24 wells depending on the availability of antibodies). But you'll need an inverted microscope for imaging, with long working distance objective lens (this depends on the stage design of the microscope).
I have some experience in photometry. According to my experience there should not be problem , if you will use plastic base. In my experiments, I used different wavelength of light with glass and plastic base both but found not significant difference in fluorescence. Also, you can try different objectives (I am not sure about your microscope) according to your need but be sure that thickness of your base should be compatible with your higher objectives. In short, there is no significant difference in result but you can also check different objectives to see its detail effect as it 's effect may also vary in different microscope.
Yes you can, the only thing you need to do is to image the plastic on its own without cells but all the mediums components inside, so you have a zero point (some media can give off some residual light) then do the same with gelatin and look at it under the microscope so you know that it does not give off any fluorescence then using another batch of gelatin add the same proportion of fluorescent antibody as you were staining cells and see how it looks under the microscope. At this point you can image your stained cells because you worked out the zero point and how the gelatin gives off the fluorescence
Thank you for all these responses. I will try the staining. Indeed I was aware that the focal length of some objectives on our confocal might be too short, but on the regular Nikon epifluo inverted microscope, that should be OK.
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