I'm working with the delivery of fluorescent-labeled nanoparticles in vivo, and I intend to measure the fluorescence of nanoparticles in tissues by confocal microscopy. However, I'm doubt how to freeze the tissues in order to preserve the fluorescence. Should I use paraformaldehyde, saline or another preservative?
Hiii Adny....
Are you making slides?? well, then you can fix it in PFA (4%) along with antifade reagent, which is commonly available.
Hi Taruxhikha, thank you for your answer. Yes, the idea is to do slices of the tissue to visualize them using confocal microscopy. However, I'm afraid I don't have time to buy antifade reagent. Do you think I can keep the tissues only in PFA 4%? Should I freeze the tissues in PFA?
Thank you a lot for your time.
Hi Adny,
Its always a good idea to use an antifade solution in your mounting medium when you are preparing slides for confocal microscopy because over longer time periods you will not see a drop in fluorescence. If getting a commercially made antifade reagent is not feasible, you can make them in yourself. Here are the recipes
http://www.ihcworld.com/_protocols/histology/aqueous_mounting_medium.htm
PFA will fix the tissue, but it can also change/increase the autofluorescence of the tissue, which may make it more difficult to find your nanoparticles. So it's a toss-up whether to use it. If you're doing frozen sections, I would be tempted to just prep your tissues in OCT. In either case, the antifade reagents mentioned above are a good bet.
Our lab has fluorescence in tissues routinely. I freeze the samples directly in OCT at -80C. After cutting frozen sections, I take the slides directly to the microscope and don't use a coverslip even but I use an epi-flluorescence microscope rather than the confocal. I have given slides to fellow lab members to image on the confocal and haven't heard any complaints.
Hi Adny..You can directly store the tissue sections in -80 without PFA. Just before imaging you can fix the tissue with 4% PFA if you want to counterstain with DAPI...it works wells.
Good Luck !!
Hoescht stain is also excellent for nuclear staining. I have come to prefer that one over DAPI.
Use n-propyl gallate as antifade dissolve a tiny bit in your mounting solution usually mowiol. Use just a few grains about 5 mg/ml is enough and the excess wont dissolve
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