I have recently discovered that a good chunk of my brain tissue has holes in it. So much so that it almost looks like swiss cheese. We have had this issue in the past and we concluded that it was because our -20 freezer was malfunctioning. Ever since then, i have been storing my brains it in our main lab (-20) freezer (which is temperature monitored) and its happened again! This time a student brains were affected as well. I would like to conclude its frost damaged once again and our main lab freezer is also malfunctioning however, I would like to rule out every possibility. Is there any other possible reason why my tissue could look like this?
The specific steps I took were:
1.) Perfusion with 1xPBS then 4%PFA in 1xPBS
2.)Fix heads with ferrules overnight in 4%PFA in 1xPBS -leave in 4 degree fridge
3.)Extract brains and leave overnight in 4%PFA in 1xPBS-leave in 4 degree fridge
4.)Transfer to 30% sucrose - left brains in sucrose for 1 week
5.)Freeze in OCT in tissue blocks at -20 (main lab freezer) - brains were left if freezer for 3/4 weeks
6.) Section on cryostat (-19 to -21) and store in 1xPBS + 0.02% azide in 4 degree fridge
step number 2 is not needed, you can extract brain and jump to step 3
Transfer the tissue to 15% sucrose and place at 4C
After a day, transfer tissue to 30% sucrose for 2 days at 4C
(one week looks too much to me)
Rinse your tissue in OCT few changes so that sucrose solution from the surface of tissue is replaced by OCT
Place your tissue in fresh OCT in Cryo block and orient the tissue (simultaneously placing the block on dry ice) and keep your cryoblock on Dry ice for 30 minutes.
Once the block gets frozen, transfer block to -70C or -86C whatever available in your lab (not at -20C)
You can leave block at -70C upto many months
Bring tissue to cryostat and equilibrate with inner temp of cryostat for 30 minutes and than start cutting
For whole brains or large tissues I would not freeze your tissue in the -20 or in dry ice as the freezing process will not be even and can result in holes and other tissue issues. As Subhash suggested, I would skip step 2 and go straight to removing the brain tissue and post fixing it overnight. Then switch to 30% sucrose as you are doing but only till the tissues sink to the bottom of the container. Once they sink, they are fully cryoprotected. Now you will place the brains in molds in OCT, however; for even freezing you want to use an isopentane (2-methylbutane) bath in liquid nitrogen. To see how this is done, please refer to this video.
https://pages.10xgenomics.com/sup-how-to-visium-tissue-prep.html?wchannelid=92q73hscm6&wmediaid=2geujjrciq
This looks like freezing damage. Freezing at -20 will cause water crystal damage because it is too slow, especially if the tissue and OCT were previously at room temperature. No matter how long you keep it in sucrose this will keep happening if the freezing protocol isn't changed. Try the isopentane bath in liquid nitrogen, you will get much faster and smoother freezing
- Badges
- Science topic
- Similar topics
- Biological Science
- Anatomy
- Tissue