I have sacrificed treated BALB/C mice and collected the pancreas and stored in -20 degree centigrade for 3 months. Now I want to isolate the islets to study the release of different cytokines from the islets. Is it possible to isolate the islets after storing for such a long time and how can I isolate the islets?
If you mean isolate and grow in culture then no. The cells needed to be isolated pre-freezing and grown in culture before being stored in storage in anti ice crystal forming agent (DMSO) supplemented nutrient media. The tissue is dead. All you can do now is make frozen sections and microdisect the islets or just do immuno staining, or Western Blotting for proteins of interest if you lyse the tissue or micodisected cells.
Rabeah Al-Temaimi Thanks for the answer. Yes I want to isolate the islets from treated mice pancreas which is kept for around 3 months at -20 degree centigrade to perform western blot, immuno staining etc and not primary culture. And should I use Ficoll/Percoll gradient to isolate the islet from stored tissue?
Well, you said study the "release" of cytokines which needs living cells! So you want to study what cytokines are "produced " by islets. If you added a cryopreservative then yes density gradient might work, but if they were just frozen as is, then no. The cells will thaw and ice crystals would have damaged the cell membranes puncturing them and as it thaws hydrolytic enzymes will degrade the tissue into a slurry. Your best option is sectioning frozen tissues and fixing them to retain cellular boundaries and doing immunostaining, or cutting small bits of pancreas where the islets are located while it is still frozen and digesting the sample into a total lysate and follow with WB.
Rabeah Al-Temaimi Thanks. For production during treatment period of mice and not release (using Western Blot), I guess I can perform Islet isolation from stored Pancreas, right?
only if it had cryoprotectant added before freezing. Otherwise as i said before, you can only make frozen sections, or whole tissue lysate.
Rabeah Al-Temaimi thanks. So for islet isolation from treated mice pancreas, I cannot store the pancreas and have to do the isolation immediately. Thanks.