Samples are fixed, embedded and frozen following a standard protocol though when sectioned at 10 um the tissue is flimsy, delicate, and missing sections.
if it is between E8 and E11, I would recommend you to fix it in 4% PFA overnight at 4 degrees/cold room. Most of the protocols mention fixation upto a few hrs. Overnight treatment with PFA makes the tissue more stable. wash once with PBS and then leave it in 20-30% sucrose (made in PBS) overnight at 4 degrees. wash again with pbs and then Embed it in OCT using dry ice. the the whole embryo at -15 degrees in the cryostat...should work fine..let me know if theres something
if it is between E8 and E11, I would recommend you to fix it in 4% PFA overnight at 4 degrees/cold room. Most of the protocols mention fixation upto a few hrs. Overnight treatment with PFA makes the tissue more stable. wash once with PBS and then leave it in 20-30% sucrose (made in PBS) overnight at 4 degrees. wash again with pbs and then Embed it in OCT using dry ice. the the whole embryo at -15 degrees in the cryostat...should work fine..let me know if theres something
Or between the last 30% sucrose and OCT you can try o/n incubation at 4C in 1part 30% sucrose and 1 part OCT. Or I quickly transfer specimen in OCT to rince out the 30% sucrose before placing them in mold to embed on dry ice rather than N2.
If I don't section the specimen the same day then I store blocks in -80C but I will let the block sit for 15 min in the cryostat before sectioning. I do 8 and 10 um. And the fixation for few hours did work for me too..
Please let us know what works for you it's good to know there is alternatives to play around.
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