I have a few human brain samples that I need to section which have been formalin fixed (10% NBF) for about 6 months than transferred into PBS w/ sodium azide for the remainder of the time until cryosectioning. Here was my procedure:
1. Remove samples from PBS and wash with distilled water
2. dried w/ kim wipe
3. embedded in OCT
4. froze on dry ice then transferred to -80C
Upon sectioning, I noticed that I am not able to get a full section but instead the tissue is cracking in different areas. Almost to a "shattering" effect as we see in paraffin tissue samples. Temperature has been adjust from -22C to -17C both with the same results.
Has anyone encountered the same problem and were able to successfully fix it?
Thank you!
Hi Isabel, your protocol is ok so far, but the temperature of the tissue block might be to cold and your cutting speed is to fast. That results is this kind of "shattering" you are talking about. I recommand block temperature around -12°C and cut very slowly. We cut our rat brains at block temoerature -11°C and chamber temperature -22°C and get good results. Try to play a littel bit with the temperature and the cutting speed and you will get good results as well. Good luck!
Hi I have some sort of similar question to this query. Is it somehow possible to perform cryosection with an intact skull?
Do you recommend to store the brains in sodium azide 0.05% and sucrose 30% for a week or two?
I would not recommand to use sodiam azide as a preservativ. We use 30% sucrose in thimerosal containig PBS (0,02 g/l), because Sodium acide could inhibid peroxidase dependened immunhistochemical reactoins (DAB-staining).
I am also wondering why my section cracking sometimes happened. But actually I use the same protocol everytime as shown below. But the result is unstable. Please help to address. THanks a lot!
My Protocol:
1. Put the brain into 4% PFA for 24 hours.
2. Transfer the brain into 30% sucrose and keep for 2 days.
3. Take out the brain and dry it completely before putting the OCT.
4. Store the block at -80oC overnight.
5. Perform cryosectioning at -25oC
Hi Isabel,
since this question seems to be still really relevant I wanted to add one small point, which has not been mentioned so far, but might be relevant.
Upon OCT embedding we usually keep samples for some time more at RT in order to allow equilibration (+ air bubble removal), before placing at -80 °C.
(However, Iam here talking of mouse brain tissue....)
Best regards,
Michi
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