I want to do cryosections of pancreas, but the tissue is really troublesome. I'm giving the animal (mouse) an injection with a hormone and following harvesting the pancreas. Before the harvest the pancreas is perfused with saline to remove the blood and any trace of unbound hormone. I've tried to snap freeze it in Isopentane and embeddet in OCT and in PFA 4% overnight followed by 30% succrose before embedding. But my sections looks like crap. I've cut them at -22 - -16 degrees.
iPentane - 70C, right?
Saline perfusion could give you some troubles during freezing.
Do you use brussh or stretcher cutting technique?
Cheers,
Ivan
After perfusion with saline, perfuse with PFA to have a better fixed tissue. (it is not critical, but can make a better fixation)
cut the pancreas into smaller parts before fixing in PFA O/N.
incubation in 30% sucrose is required, the tissue should sink to the bottom of the container which indicate complete equilibrium of sucrose.
Incubation of OCT before embedding, at least 1 hour or even O/N in 4 C. This step again make sure that the tissue has good infiltration of OCT.
While cutting, use -14 C. To cold will make tissue to hard and crack easily.
This should enable you to cut at 5-10 um. But people like 14um. You may also try 20um if the tissue is really hard to handle.
Hello Emilie.
I totally agree with Ka Kui Tong.
Below you can see our protocol. Maybe it helps:
1. PBS-perfusion (5 min)
2. 4% PFA-perfusion (5-10min)3. Postfixation in 4% PFA (ON)
4. Wash in PBS (ON)
5. Cryoprotection in 30% sucrose (till it sinks to the bottom)
6. Freeze in Isopentane (5 min?)
7. Store until cut in -30°C
8. Embed in OCT
9. Cut
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