Hello everyone, I need advice on improving the quality of my coronal slices obtained from PFA-fixed mouse brains using a cryostat. As You can see in the attached image, slices are horrible! Any insights on the potential issues would be greatly appreciated.
The brains are in 30% sucrose. I cut them into 4 mm slices. These slices were then frozen using the immersion of the cryomold in acetone left at -80°C (i did not have dry ice). The samples were then stored at -80°C for one day. The cryostat was set at -20°C, and samples were left in the machine for an hour before slicing. The slice thickness was 20 μm.
After cutting, I touched the slice with a glass slide and allowed it to dry for 20 minutes at room temperature to prevent detachment. Subsequently, I washed the samples with PBS and performed Hematoxylin staining. After drying, the slide was mounted with Entellan. Any comments or suggestions would be invaluable as I'm currently facing challenges in this process.
Hey Guillermo
I was facing the same issue when sectioning mouse-liver. I got rid of these fractures playing around with the temperatures and actually increasing it to -10°.
Also maybe check if your blade is really new and sharp, this does huge differences, at least for livertissue...
Here I found some Troubleshooting:
https://www.linkedin.com/pulse/20141009215234-99751721-the-basics-of-the-dreaded-cryostat
Hi Guillermo C. Fernández
Is it happening post sectioning? or during the staining steps?
* If you observe the same slides post drying for an hr in microscope after sectioning and if the vacuoles or fractures appear it may be due to improper fixation or sucrose preservation.
* If the same are observed during the staining steps, I would suggest you to dry the slides for an hour before use, fix the slides in ice-cold methanol for 10mins and proceed with the staining.
Hope it works.
Thanks,
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