Hello, I have a problem with staining the mouse brain.
These days, my professor told me
"There is something wrong with your images. I think you must have made the mistake in perfusion or fixation. It's not compact. There are too many holes"
But I can't fix the problem.
Could you help me, please?
There are vacancies around the nucleus like edema in H&E and IF images.
I think it is just a nucleus hole.
There are expansion of clear spaces around the neurons, and given the diffuse distribution along the lack of significant lesions, the changes you see is likely artefactual from fixation issue eg. delayed fixation, concentration of fixative etc. Once the tissue is fixed and processed, there is no way to reverse these changes.
Hi Kyungri Kim
If these are cryotome sections, it may occur due to improper cryopreservation (10-30% Gradient sucrose) or it can be due to differences in the cryotome inside temperature and the sample holder temperature. I would advise you to optimise the temp, use a new blade and check the sections in microscope before staining. If you can observe it prior to staining then the same can be confirmed.
Thanks,
On the contrary, could you please proceed with a new section, this time adhering to the following steps:
In conclusion, it's crucial to recognize that a uniform lab procedure may not be suitable for all tissues; the brain, for instance, cannot be treated the same as other organs. The thickness of sections should vary, considering that brain tissue is less resilient compared to other tissues. Experience plays a significant role in optimizing and adjusting lab protocols. If you encounter challenges that cannot be rectified, as you're all thinking it's a fixation error, consider taking your block to a nearby histo lab. Their expertise can help produce a well-stained outcome. Since we cannot visually inspect the issue, our assistance is limited. But I doubt it's a fixation error!
However, unless you confirm challenges like brittleness or difficulty obtaining a section or ribbon during sectioning, I can tell you it's likely not a fixation error. Your nearby histo lab remains a valuable solution for resolving such issues.
Sincerely, your nearby histo lab is your solution arena, consider taking your block there for help...
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