Hi,
I am doing a research on the effects of lead and magnesium on NR2B receptors on mouse hippocampus. Does anyone have a protocol how can I preserve/ freeze hippocampus tissue? After I collect all the samples I intend to homogenize and run the ELISA test for NR2B concentration in the samples.
Best regards
You should flash-freeze it (in liquid nitrogen for example) and keep at -80.
We perfuse mice with 20ml ice-cold PBS (to remove blood from the brain), place dissected hippocampi into Eppendorf tubes and place tubes into liquid nitrogen. Store at -80.
Instead of liquid nitrogen, you can use dry ice mixed with 80% Ethanol. It also freezes fast but does not evaporate as quickly. Make sure you use permanent marker to avoid your labels being washed away by Ethanol.
Perfusion of animals is NOT recomended if you need to estimate native concentration of protein like NR2B by Elisa reader, just wash tissue with saline solution and plot on filter paper and rapidly freezing
Sounds like since you are not trying to do histology flash freezing is probably just fine. I have been told perfusion etc. is much better for histological staining in order to prevent ice-crystal accumulation. Hope this helps!!!!
I also believe perfusion is not needed if IHC is not the one you are looking for. Moreover to do perfusion you have to give the animal a deep anaesthesia.....and that may disturb the actual scenario of the brain regarding your protein of interest. In addition, the delay in freezing the tissues will definitely give you more unwanted post-mortal changes. Thus after cervical dislocation method one should try to dissect hippocampus ASAP and flash-freez it in liquid nitrogen before store at -80 degree C until use.
Hope it helps and all the best for your experiment.
I apologize for confusing everybody about perfusion and appreciate opportunity to learn from my colleagues.
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