frozen sections - (Apr/15/2005 )
Hello I need some help with frozen sections. Basically I am harvesting mouse embryos out of the yolk sac, embedding them in Neg 50, and putting them directly into the -80 freezer. To cut, I warm them to -20, mount, and cut.
This procedure has worked beautifully for the older embryos (E14.5, E15.5); however, I just cut some younger embryos (E12.5-E8.5) and their morphology was horrible. Not even organ structure is distinguishable! Yet I did not do anything different.
Does anyone have any thoughts on what the problem might be?
Note: the E9.5, E8.5 I usually leave in the yolk sac since they are so small.
I have worked with many different ages of mouse embryo before primarily using their sections for radioactive In Situ Hybridization. Cryosectioning is not a problem provided that the embryos are paraformaldehyde fixed (4%, 4-24hrs), cryoprotected (10-20% sucrose in PBS o/n is okay), then embedded in OCT (using a methanol/dry ice bath) and store at -80.
If anything, I have had a hard time with sections from the older mouse embryos (E17 or so) because the fix, cryoprotectant, and OCT dont penetrate as well.
REmerson - To me it sounds like there may be two possibilities:
(1) It sounds like the samples are not fixed before freezing? If so (and I often choose this type of specimen prep myself) this can lead to a deterioration in tissue morphology after frozen sectioning although it still is possible to get great sections from fresh frozens but you need to fix before staining with something (if doing H&Es use formalin, if doing IHC you may want to tes acetone as well as formalin). Otherwise you can fix before freezing although expect some antigens to not be well preserved if doing IHC.
(2) But more importantly it seems as if perhaps you are getting a freeze artifact. Generally speaking putting samples in embedding medium directly into the -80 deg C is not considered a fast enough freeze. You ideally need to submerge in isopentane cooled in a vessel in liquid nitrogen for FLASH frozens. I expect this will help dramatically.