Dear Researchers,
I am working on an animal study and I want to produce high quality of tissue imaging. The probelm, is that I have low quality of cryosectioning which can be noticed after tissue imaging.
Current Lab Protocol:
Harvest the brain without perfusion.
Freeze it directly on DRY ICE, without Liquid nitrogen or Cooled Isopentane.
Freeze at -80.
Cryosection at -16 and champer tempreture -20 degree (12um sections) with Leica CM1850 Cryostat .
Any ideas?
Thank you!
Best Mohammed AHMED.
Hello,
I am curious why you are not perfusing. Blood left in the brain tissue may interfere with antibody staining and give non-specific background. If you perfuse with PBS followed by PFA (and let fix overnight in PFA) tissue may hold up better. Use 30% sucrose as a cryoprotectant (to prevent crystallization) and move to sectioning (slow freeze in -20 overnight). You are not really getting a uniform freeze just placing your brains directly on dry ice. 12um sections are very thin--- I don't cut brains below 30um in a cryostat (not saying it isn't possible, it's just difficult). For thinner sections (5-10um), paraffin embedding is the easiest way to go.
If you need clarification, please let me know!
I hope this helps!
Elliot
Why are you freezing it? Are you doing IHC or other staining such as lipid or EHC, which needs frozen tissue sections? Can you not just do FFPE on the CNS?
You get artefacts with both FFPE and frozen. Essentially all histology is an "artefact" that you hopefully minimise ;-).. Do you have a JPeg?
Dear Mohammed,
I also suggest that you share images of your sectioning artifacts. That way the RG community can offer better tips. Also, what type of imaging are you trying to accomplish?
I have successfully frozen rat and mouse brains on dry ice and performed cryosectioning. However, the sections were fixed after that with a fixative that was compatible with antigen preservation as specified for the primary antibody I needed to use. It can be a bit tricky to get the powdered dry ice sifted onto the brain without deforming it. You must carefully surround the brain with the dry ice--you can't just set the brain on the aluminum foil on a big chunk of the dry ice. For that reason I suggest that you perfuse your animals as suggested by Elliot. Neurons die quickly, and perfusion gets the fixative deep into the brain rapidly.
Even if you decide to cryosection the fixed brain, I find that I need to play around with the temperature a bit from day to day (plus or minus a degree) to get good sections (I usually cut 11 um sections). You also need to let the tissue warm up to the temperature of the cryostat before you section. If you try to section tissue that is too cold, the sections will shatter.
Best,
Jill