Hi everyone I am running ETC for clearing mouse brains. I am not sure if one can reuse the pre-used SDS buffer (7-8Litres used to clear 1 mouse brain 30V/37oC) for clearing another couple of brains or one has to use fresh buffer every time when clearing brain. Though the pH of the used buffer is fairly constant, but I am not sure how one can now if the buffer is saturated with lipids. Re-use of SDS buffer will lower down the running cost. Any suggestions are most welcome ?? thanks
I use the SDS clearing solution until the pH drops down to 7. I also have about 7 liter in the entire system and run two chambers at a time. It takes about 10-14 days until the pH is at 7 (staring at 8.5). In addition, I highly doubt that a few little mouse brains will saturate your clearing solution to a degree that there is significantly less clearing. I would just go by the pH.
thank you very much for your suggestion, I will keep an eye on pH. Even after clearing brain for 8 days (25V/37oC) i could not make it to transparent. Its a yellow mass, with partial transparency. Would one run ETC to extended time period. any thoughts ?
Wash with PBST then incubate in FocusClear.
If the brain is clear then wash again with PBST and continue with antibody staining.
If its not clear then wash again with PBS, then change to clearing buffer and continue clearing.