Please see the attachment.
In my case, I tried a lot of time on the Nissl staining on 10um cryosection. I am not sure which step goes wrong? I make sure my anti-rolling glass is intact. seem like it's still cracking?
Thanks for help. I appreciate!!!!
My 30% sucrose in 0.1M Phosphate buffer recipe:
- mix 150mL 0.2M Phosphate buffer (pH 7.4) and 90g sucrose
- Fill Milli-Q water to 300mL
- Mix well and filter it
- Store at 4oC before use.
My protocol is below:
1. Mice are perfused with 4% PFA
2. Post-fixation of 4% PFA for 24 hours
3. Replaced with 30% sucrose (made with 0.1M phosphate buffer pH 7.4 and make up with MiliQ)
4. Keep in 30% sucrose for 2 weeks (replaced with new 30% sucrose in between for once)
3. before doing sectioning, make the block with OCT, and then put the block on ice bath for 10min, and then transfer it onto dry ice block for 15min (let it to be freezed slowly)
4. After that, put into the cryostat
5. Cut the section in the crytostat under -25oC
Protocol of crystal violet staining of postnatal mouse brain
Prepare 50mL 0.1% Crystal Violet solution
- 0.05g crestal violet acetate (Sigma, C5042)
- Make up to 50mL with Distilled Milli-Q water (MQ)
- Sonicate for 15-20min for completely dissolve
- Add 125uL acetic acid to the 50mL solution
- Filtered with Whattman filter paper
Staining protocol
1. Take out the cryosection from -80oC and place at -20oC for 20min
2. Take out the cryosection from -20oC and place at 4oC for 20min
3. Take out the cryosection from 4oC and place at RT for 20min
4. Pre-warm the 0.1% crystal violet solution at 37 oC
5. Wash the slides with xylene for 5min
6. Wash the slides with 95% ethanol for 3 min
7. Wash the slides with 70% ethanol for 3 min
8. Wash the slides with MQ for 3 min
9. Stain the slides with 0.1% crystal violet solution for 10 min
10. Rinse the slides with MQ using dropper
11. Wash the slides with MQ for 3 min
12. Wash the slides with 70% ethanol for 3 min
13. Wash the slides with 95% ethanol for maximum 1.5-2 min
14. Dip the slides with 100% ethanol for 2-3 times
15. Wash the slides with xylene for 5min
16. Mount the slides with Permount
I’ve found, that the fewer steps there are, the less dehydration, the less cracking occurs. Have you tried any of the aqueous permanent mounting media? There are many brands. Example: https://www.bio-rad-antibodies.com/reagent/aqueous-mounting-medium-accessory-reagent-buf058.html?f=ready%20to%20use&thirdPartyCookieEnabled=false
Hello,
it looks like that there are a lot of freezing/thawing artefacts.
When cutting the brain, the glass slide should be allways inside the cryostat and only the site were you want to place the next tissue slide should be "heated" with your finger tip (Some institutes have a small heat plate inside the cryostat. The plate is very small, like a 50 cent pice) and then you place the tissue slide on the heated spot, where the slide will then be fixed.
If you allways take out the glass slide from the cryostat (about 6 times, with seven tissue slides) and put it again inside for the next slide, you will have a lot of freezing and thawing artefacts.
Kind regards
Stefan
This are cryo artefacts. I agree with Stefan, but in my opinion the reason therefore is not the transfer onto the slide . The reason is the deep freezing process before cutting. You should feeze the block as fast as possible to prevent cristal building in the cells. This cristals disturb the cell structure. After deep freezing in drye ice the tisssue block should equilibrated in the cryostate chamber . Start sectioning very carfully. Play with chamber and block temperarture to find out the optimal temperatur for sectioning . It veries form tissue to tissue.
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