I have stored my mouse brain samples in liquid nitrogen just after taking from anesthetized mice. After that I stored them at -80c for prolonged storage. Actually I did not put my samples in OTC nor in 4% formaldehyde as many researchers suggest. Now I have to make a paraffin embedded section from these stored brain (each section cortex, hippo-campus, thalamus separately). Kindly suggest a suitable method to get section and the preferable size of sections.
You will not have many distinguishable histologic features if you embed snap frozen tissue...almost everything will be freezing artifact. If you are planning to do H&E or even IHC staining and scoring, very likely it will be uninterpretable.
Unfortunately I must agree with Pawel having tried to do this myself. I found poor preservation of features and uneven staining.
I must agree with Pawel's and Brian's statements above, but that doesn't necessarily mean that the tissue is useless though. You may have to change the techniques and approach that you take with analyzing this tissue. I am not sure which markers, proteins, or hallmarks you might wish to probe for, so this advice may not fully apply.. Based on the way the tissue was flash frozen and stored, the only thing you really can do with it is homogenize the tissue with a tissue lysis buffer (with protease inhibitors), and use the lysate in alternative assays such as western blotting and/or ELISA. While you will not be able to visualize the tissue through microscopy, you will still be able to observe possible changes in protein levels between groups, using these techniques.
Thanks everyone. Yes I agree for other analysis such as mRNA quantification, western blotting or ELISA i am sure i will be able to get expected results anytime. I was just curious about histological studies.
Hi,
I would aggree to use that only for biochemistry experiments now. But if you want to do morphology studies, H&E, IF, then you better section the different brain parts before PFA fixation. I would also suggest to carefully freeze the isolated parts in tissue freezing media in isobutanol (cooled down to a milky colour with liquid N2).
Best,
Jonas
- Badges
- Science topic