We are trying to encourage quick lipophilic dye uptake and transport in unfixed brains - we are using ones that were flash-frozen immediately after sacrifice and were then quick-thawed, though I realize fresh unfixed tissue would be better for morphology once we can get those. We are avoiding fixation as we have read that it dramatically slows the dye transport. Also, increased temperature encourages faster dye diffusion through cell membranes. Thus, I have one such brain with dye added, being kept in PBS with 0.05% azide at room temperature to encourage dye transport while keeping the brain hydrated and preventing microbial growth.
How long can unfixed (brain) tissue last in PBS with azide, either at room temp or at 4°C? I realize azide may not prevent everything from growing eventually, and I am unsure how quickly proteases may act within/on the tissue. As far as what I mean by the tissue "lasting" or "degrading", I am looking to retain just general whole-cell morphology for fluorescence microscopy without much concern about cellular parts like mitrochondria, nuclei, or dendrites. The freeze-thaw I mentioned earlier likely disrupts the morphology, but I wanted to see how much our particular storage condition may do this as well. Thank you in advance for your time and input.
The quick answer is: Not very long!
Well, neurons are supposed to be dead by 3 mins without glucose or oxygen. PBS with azide is not at all a good buffer to use. Azide is a respiratory poison.
So, my suggestion would be you should use sterile physiological buffers that preserve the cells. A good idea would be to put them in normal tissue culture media with bi-carbonate buffer or use sterile Hank's balanced salt solution without azide. But even with these you can not preserve them for long. Try to finish your experiments as early as possible.
Regards
Sodium azide is to prevent bacterial actions but it will not prevent cellular metabolism like apoptosis without medium and from other endogenous degradation. We normally use sodium azide in antibodies or some reagents to preserve where there is no cellular activity. In short, not a good idea
Thanks for your inputs. Although I'm not concerned with tissue viability as the dye can be taken up and diffuses passively, I was unsure how fast cellular mechanisms of degradation would occur even in the absence of microbial decomposition. I underestimated apoptosis and the speed of caspases, it seems.
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