Incubation time in SDS clearing solution

lijun

The time I used to cleared the brain with ETC ( 1A,25V,33°C) is about 9 days,it seems much longer than other people(2~3days).

I'd like to know if a longer incubation in SDS clearing solution will cause any damages in samples or a better clarified performance?

lijun

one more question : if the pH of PBST that used in washing the SDS clearing solution and the storage of the embeded sample should be adjust to 8.5 ?

Kristin_Engberg

Clearing time with ETC can depend a lot on your specific ETC set up and settings (voltage, temperature, flow rate). 9 days is not unreasonable for good clearing. Longer incubation in SDS should not damage the sample (to passively clear, the samples sit for 6-8 weeks at least in the clearing solution), and the longer the sample spends in the SDS buffer, the clearer it should be. In our lab, we often use ETC for a few days to take care of the majority of clearing and then passively clear it for a week or two to get well-clarified samples.

The pH of the PBST does not need to be adjusted to 8.5.

DavidLenzi

So what's the best approach to knowing when to stop electrophoresis? Can one stop, transfer the tissue to PBS, then FocuClear, check transparency, then if needed go back to PBS, clearing solution, then back to electrophoresis?

yetiti

Hi DavidLenzi, Yes you can definitely do that. Make sure you wash out the SDS (really) thoroughly.