When to change SDS buffer while running ETC

Shashank20

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

DominicJG

Hi Shashank20,

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.

Shashank20

Hi @DominicJG,
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 ?

Fabio

Your brain will become fully transparent after you put it in FocusClear, not after ETC

Shashank20

@ Fabio, thanks for your reply. Actually I am trying whole adult brain imaging and I plan to proceed to antibody staining step, before going to Focus Clear. I am worried if the lipids are not cleared totally (as the sample is partially transparent or translucent) , I might land up in poor antibody permeability and huge autofluorescence ? any comments ? thanks

Fabio

If you want to check how clear it is:

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.

Vortexam

Dear all,i have problems in dissolving add in my clearing solution.I used SDS pellets,initially i made oné litre solution.Even after adding SDS slowly,solution remains turbid

Fabio

If SDS dont dissolve it can be a contamination in the batch. Just try another one. Also dont do your solution at temperature lower than 20 °C because your SDS will precipitate.