I am trying to do some immunofluorescence on mouse testis sections but after staining and mouting, the tissue seems to degrade rapidly and becomes smudgy, in particular nuclei. Has anyone experienced this and managed to fix it?
My protocol is:
- Harvest testes, pierce tunica, immerse in OCT and snap-freeze.
- Cut 5-8 microns sections on cryostat (temperature -20C - -18C), mount on Superfrost plus slides and let dry on plates at 37C for 30min.
- Keep at -80C until staining.
- For staining slides are let dry for 20min, rehydrated with PBS for 5min, blocked and stained with primary/secondary sequenntially. I do not include a fixation or permeabilisation agent.
- Mount with Vectashield+DAPI and add coverslip before imaging.
I had tried to fix my tissue (4% FA at 4C overnight followed by sucrose) before freezing, but the qquick degradation still happened.
Thanks for your help!
Hello Remi,
to solve this problem you have to perfuse your mice, because the duration of difusion takes to long.
Or instead of immersion fixation deep freez the tissue imedeatly after sacrefaizing the animals. Cryosectioning; after transfer on the slide just dry the sections until they become transparent, put the slides into a small slide box which you have placed in the cryostat. Deepfreez them at -80°C until you start your immuno. Before you start your immuno adjust the sections at room temperature. Then put them in 4%PFA at 4°C overnight . Next day rinse shortly in distilled water, 3 x 5 min PBS, 2 rinses in 70% ETOH, 100% ETOH 20 min at 4°C, 2 rinses in 70% ETOH, 3 x 5 min PBS. Continue with your immuno protocol, If you don't see any specific staining in your sections then you have to perfuse the mice because of the degradation.
Hello Remi,
I just started perform IF for frozen tissue (skeletal muscle and skin) a few month ago. I don't have any problem with degradation as yours. Here come the protocol of my lab: gelatin - coated slides; after sectioning (7 microns), let naturally dry up in room temperature about 15-30 minutes, fix in 2% PFA at 4°C in 10-20 minutes, wash 3 times with PBS 1x in 4°C, let dry in room temperature (about 10-30minutes), then freeze -80°C until staining. One other thing, we use OCT and isopentane to make the cryo-blocks, and the tissue size is quite small, about 6x6mm2 in maximum. Hope it helps.
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