So I am new to cryosection and have been following a published paper's method where heads are fixed OV in 4% pfa in PBS 7.4 pH at 4C, then transferred to 20% EDTA and 10% sucrose OV at 4C and then 20% EDTA and 20% sucrose OV at 4C. I deviate at this point and use OTC to embed the tissue, the paper uses gelatin and sucrose, and flash freeze on dry ice and then for 2 hours at -80. I am trying to get 14 um sections but am having great difficulty getting consistent slices. Most of the time the blade slices the block nice and clean, no chattering, and when it gets to the tissue it melts through it or crumples up the tissue not the section though. I have tried to rub a thumb on the specimen, and also remove the chuck and put it on dry ice for several minutes and go back to cutting, this led to some okay slices but nothing great and beautiful like the paper. Again, I am new so any advice would be greatly appreciated! Pictures of my slices provided.
Hi Greg,
I did cryosections some years ago and I can recall the same problem as you are describing. I was working with different human tissues, and each one required different ambient and blade temperatures. If it was not the correct one, the tissue wouldn't come out along with the OCT as you have shown on your pictures. So, I would suggest to try to find the optimal temperature inside the cryostat and of the blade.
Hope this can be helpful,
Cheers,
Daniel
Hi Greg,
I had also faced same problem but sorted out by increasing the Coarse feeding speed of microtome during sectioning and you can also try to maintain temperature inside the freezing chamber.
Good Luck!!
Shambhu
You may already do this, but if not, make sure you allow the block to equilibrate with the cryostat temperature. Let it sit in the chamber for ~15-30 minutes prior to mounting for sectioning. It may be shriveling because of an abrupt temperature change between your tissue and the cryostat metal.
So I tried acclimating the block to the chamber and also am using a freezing spray to cool the blade and block if it warms too much. The results are slightly better but still some cracking of the sample. I read so other similar questions and see some say overfixing can cause cracking, do you have any experience with this? I am thinking of fixing a head in 2% pfa OV to see if this helps, or would fixing in 4% for 30 min at RT be better? Thank you all for all your help, time, and suggestions!
I've cryosectioned intestinal segments, so there may be differences. We would fix 1-2 hours in 4% PFA at room temperature, rather than overnight. Also, some times slicing through slowly helps preserve the tissue, even though swift slices are recommended.
You might try not to fix and cryoprotect your heads but immediately embed it in OCT medium, flash freeze on dry ice and cut then, without any more steps. In this case incubate with your primary antibody without any retrieval steps. I'm not sure about blocking for endogenous peroxidase is necessary if using fish tissue and HRP-labelled secondaries.
How old are your fish? It seems like they are at least several weeks. At this point the skull is quite thick and hard and will be quite difficult so section with cryosections. You can try a decalcification protocol or maybe there is a digestion for cartilage? was the gelatin embedding used with heads, too? It might freeze a bit harder and work better. For such hard structures plastic like JB4 plus works quite well, but you can’t do antibody staining on plastic and would have to do this before embedding.
The fish are a year old, so yes the skull is as thick and hard, that is why I decalcifiy in 20% EDTA for two nights, this has been shown to soften the bone and cartilage for cryosectioning in the past. I found another protocol using agarose-sucrose to embed and freeze in liquid nitrogen before sectioning. I am going to try the non-fixed method, as well as try 2% and 4% pfa at RT and OV to see if over fixing is an issue. Thank you again for all your help!
Just a quick advice: Due to the Leidenfrost effect it's not recommended to freeze directely in liquid nitrogen, because it gives you a "warm" vapour of gaseous nitrogen which isolates the organ from freezing. It's better to cool isopentane (2-methyl-butane) in liquid nitrogen and freeze the fish in this. It's cold enough when the isoopentane starts to solidify at the bottom of a metal pot.
And try unfixed freezing after the EDTA step, it should work.
Hi Greg,
It's good to find your question here since I am having the exact same problem! I also fixed the heads in 4% PFA ON at 4C, then the cryopreservation+decalcification step with 20%sucrose, 20%EDTA and then embedded in OCT in dry ice on a metal plate. And the sections are "empty" as in your pictures. I first thought that there was a problem with EDTA (not enough time or low concentration) but the skulls seem soft to me after EDTA so maybe is the fixation time as someone pointed out???
Have you succeed in any of the protocols?
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