I want to do in vivo injections of RITC (rhodamine-b-isothiocyanate) into the eye and PHAL injections into another brain structure, followed by immunohistochemistry with anti-PHAL antibody and a fluorescent secondary antibody.
Will the RITC labeling persist through the immunohistochemistry procedures? I am worried that the 0.3% Triton used together with the antibodies may extract the RITC from the tissue. Would the immuno also work without Triton, perhaps? Or are my doubts just unnecessary?
Another possibility would of course be injecting CTB into the eye instead of RITC, and doing different immunofluorescence via anti-CTB and anti-PHAL. But RITC is cheaper and requires less antibody usage, so I want to know if it works.
The RITC should not diffuse or diminish in signal. According to one paper, it is "resistant to histological fixation procedures". (linked below). In that study they used parafin sections, ethanol, and xylenes, which tend to strip away most lipids and thus permeabilize cell membranes..
In non-parafin tissue, fixation already permeabilizes cells to some degree already, while a detergent (Triton) increases this permeability. If the fluorescent tracer can survive fixation and de-parafinization, then it should be able to resist leakage from low amounts of detergent on the order of what you are using. In case the signal does diminish due to some leakage, you can amplify the RITC signal using an anti-rhodamine antibody and a secondary with similar fluorescence ex/em spectrums.
If the leakage does occur, your could use a similar fluoro-tracer that anchors to the inside of cells better, like RITC-dextran or fluororuby (both with MW of 3000 or less). As long as you get the lysine-fixable version of those, they will resist most IHC processing procedures. MW 3000 is important because they go preferentially retrograde, which seems like what you want, while larger ones (MW 10,000) go anterograde preferentially. The only trade-off is that these compounds are somewhat more expensive than unconjugated RITC, depending how much you are using.
http://www.ncbi.nlm.nih.gov/pubmed/6643714