I think about this idea for the sake of minimizing post-decapitation changes which take place in brain tissue in ordinary procedure in which it takes some minutes, at least, to take the brain out of the skull.
But I'm not sure if the brain could be detached from its surrounding when head becomes snap frozen in liquid nitrogen.
Hi,
well first it depends what use you plan on doing, if you want to perform histology/immunohistochemistry etc, putting the brain straight into LN2 is going to create freezing artefacts.
You have a few options:
- optimize/improve your harvest time and make sure to dissect on ice
- use PFA transfusion to fix your brain before harvest
Thanks @Marie Mortreux for answering,
I need rat brain for gene expression evaluation in the RNA levels. I know that mainstream methedology is to take the brain out then snap freezing it on dry ice or through isopentane bath in liquid nitrogen, but it takes 4 minutes for me, at best, to detach brain from the decapitated head which seems a long time without blood supplement which in turn results in inaccurate results in comparison with a living brain. I am wondering if it's possible to prevent post-decapitation changes by snap freezing whole head with fur and everything right after decapitation and proceed the frozen brain collection later on.
I understand your predicament. However, surely you are not doing gene expression on "living brains" as controls. Even the controls need to be harvested. I think if you make sure your conditions are similar, you could totally compare the gene expression profiles, it is what is traditionally done.
You could also take half your brain for western blot analysis (as gene expression is not always associated with protein synthesis especially in short time frames)
Thanks again for your attention to the subject.
Actually I'm going to isolate mPFC after treating 12 rats with psilocybin and extract the RNA content of the tissue and do RNA-SEQ to evaluate expression changes induced by this drug in comparison with salin-treated group of rats. My concern is that the time period from decapitation to freezing collected brain, exerts changes to the RNA content of the mPFC which is not induced by psilocybin but resulted from a dead brain situation like hypoxia. The traditional methodology has ignored the effect of this factor and relate the gene expression changes to the drug of interest while it might be, at least to some extent, due to a dead brain situation.