The situation is: mice brain samples were put into the -80C without adding any anti-freezing agents (e.g. glycerol, DMSO, etc) after harvesting them.

If laser dissection is not available in the lab, what else would be the option (for isolating neurons from non-neuronal cells in the whole brain scale)?

Thank you very much once again!

I assume you mean non-viable cell isolation. We thaw the tissue in formalin solution to fix the tissue as it thaws and then use collagenase (after washing the formalin fixed tissue) to dissociate the tissue into individual cells. We do this in cardiac tissues and I don't know if this will work for you or not.

All methods discussed here are helpful but first you keep your experimental tissue into -20 dgr C for a while then 4 dgr C and then thaw in the solution you used to do the experiment. One question damage in what aspect? Due to some treatment or frozen condition, because if proteins in the tissue are damaged then your experiment will be in trouble to detect what you would like to be. Better to put tissue or cell with protease inhibitor whenever you will preserved it for research work.

Wish you the best

Thank you very much for all the useful tips.

By the way, I was wondering whether the following might work in practical/reality:

1) I thaw the already-frozen-mice-brain in 4% paraformaldehyde (as inspired by Michael) through a graded thawing process (as suggested by Sarmishtha).

2) Then, I add trysin (a well-known cell isolator used commonly in cell cultures) to my sample to isolate all cells (maybe very time taking).

3) Afterwards, I add the relevant antibodies to my sample to separate different cell types apart.

4) Once I had transfered these different cell fractions into individual containers, I then start examining their protein expression profile or whatsoever thereafter.

Thank you very much once again in advance!

I do not know about the other steps, but step 2) should definitely not be done using trypsin: as the cells in your tissue may already be damaged, this protease is likely to fragment your proteins of interest (including intracellular ones) as well, and any downstream procedure (e.g. using antibodies) will be useless (and the antibodies will be attacked by the trypsin themselves). Using Collagenase, as suggested by Michael Watson, should be your method of choice.

You should also think about potential problems arising when doing protein analyses after fixation with formaldehyde ...

I am not an expert in the field but maybe this paper may help:

Acta Neuropathol Commun. 2014 Jan 7;2(1):4. doi: 10.1186/2051-5960-2-4.

Generation of iPSC lines from archived non-cryoprotected biobanked dura mater.

Sproul AA, Vensand LB, Dusenberry CR, Jacob S, Vonsattel JP, Paull DJ, Shelanski ML, Crary JF, Noggle SA.

http://www.ncbi.nlm.nih.gov/pubmed/24398250

I have a similar question, but I need to know if I can use collagenese to digest the tissue (testes) and then do a cell purification with percoll gradiente?