Hello everyone,
We are extracting total brain tissues and cutting sections via freezing stage microtome. Total brain tissues are fixed in 20 % paraformaldehyde + sucrose for 48 h and then transferred to %20 PBS sucrose. Until now, we were storing the slices at -20 degree in long term storage buffer. However, after staining some slices we got images with some kind of dirt and disruptions. The protocol for the long term storage buffer doesn't involve any anti-bacterial or anti-fungal but not all sections have this problem so we are suspecting that it is due to the storage time.
I would like to know what are the optimal conditions for storing the brain slices for long term or can we use another solution for long term storage of the sections? And how long can a brain tissue and a brain slice can be stored?
Thank you
Dear İrem,
we use this solution to store brain slices at -20 for at least several months: 30% ethylene glycol, 25% glycerin in TBS or PBS. Try it if you use something different - we never had any problems.
If you suspect that any other step of the brain fixation procedure may be the reason of your problem, here is a full description of how we do it (taken from Supplementary material of this paper Article Pro-neurogenic, Memory-Enhancing and Anti-stress Effects of ...
):After exposure to their respective compounds and behavioural testing, the mice were sacrificed under isoflurane anaesthesia. Eight mice per group were used for the immunohistochemically assay. The brains were dissected, placed in 4% paraformaldehyde in PBS (phosphate buffered saline (Lonza, Belgium); 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.5) for 24h, treated in 30 to 20% sucrose gradient in distilled water at +4 °С for 24 h, then frozen in precooled isopentane at -70°С (on dry ice) for 2 min. Brains were stored at -80˚C until 50μm thick free-floating frontal sections were obtained using a microtome/cryostat (Leica CM1950, Leica Biosystems, Germany) at -20°C. Every 8th section was taken from each brain along the proximal-distal axis, collected in TBS, then added to anti-freeze solution (30% ethylene glycol, 25% glycerin in tris-buffered saline (TBS, 50 mM Tris, 150 mM NaCl, pH 7.5) and stored at -20°C.
Best wishes,
Alexander
Dear Alexander Trofimov , thank you for the information you provided.
I was wondering if it is crucial to replace the storage solution periodically?
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