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 usually don't cut brains below 30um in a cryostat (not saying it isn't possible, it's just difficult). For thinner sections, tt requires a uniform freeze with no crystallization within the tissue as well as a properly aligned cryostat. I have recently had success with fresh flash frozen brains at 15uM. 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
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 usually don't cut brains below 30um in a cryostat (not saying it isn't possible, it's just difficult). For thinner sections, tt requires a uniform freeze with no crystallization within the tissue as well as a properly aligned cryostat. I have recently had success with fresh flash frozen brains at 15uM. 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?
- Badges
- Science topic
- Similar topics
- Biological Science
- Histology