I apologize if my question seems to naive, but I would like to do a protocol to determine bio-distribution of a peptide coupled to TMR in an animal model however, I am having some difficulties in find a protocol, and I don´t know anyone working with this fluorescent tag. I read several papers however, the protocols sometimes are confuse and not fully detailed and I have some doubts. The ideal was a protocol to use with imunohistochemistry, is it possible? Do the peptide in the organs keep the fluorescence after fixation of the tissues?
Thank you very much!
I think there is a good chance that tetramethylrhodamine will retain its fluorescence after formaldehyde or glutaraldehyde fixation because fixation involves crosslinking of primary amines, and TMR lacks primary amines. The peptides must contain the primary amines so that they will be fixed in place.
Be careful to minimize exposure of the tissue slices to light, which will gradually cause bleaching of the TMR fluorophore.
The tissue will probably exhibit green autofluorescence, which will result in a background level of fluorescence in the TMR channel, so you should also make some unlabeled samples for comparison. This background can be minimized by using a fluorescent dye with a longer excitation wavelength than TMR.
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