Tissue Storage after clearing

balsorjl

Can anyone recommend a better tissue storage technique for after clearing than PBS or PBS-T at room temp? I find the sample continues to swell and the hydrogel collapses when left in this solution for too long.

Tully

I tried to store them at a lower temperature (had them at 37C for a while), because the protocol says you can keep them in the clearing solution as long as its room temperature or lower. Which I interpreted as "refrigerator is OK". Apparently it REALLY ISN'T... It freezes to a block of ice. Does anyone have ANY idea why this happens? I had a tube of water next to it, and it didn't freeze at all. But all the tubes with clearing solution were rock solid, and basically borked all my samples.

My stained samples are stored in RIMS (refractory index matching solution), which seems to work just fine, without any notable swelling (they actually shrink after I transfer them from PBST to RIMS). So far anyway. It's pretty simple to make, and works really well for imaging.

Fabio

Dont`t put samples that are in clearing solution in temperatures below RT. If you do this the SDS will precipitate. If you want to store the samples PBST is good, for long-term 4° PBST + diluted azide works fine for me

Tully

Fabio: Yeah, I noticed that a little too late.. It didnt even look percipitated, it was like the entire tube was a solid block of ice.

Somebody should update the wiki on that.. "RT or lower" is written next to clearing solution storage.

rchurt

@Tully‌ I'd guess that the SDS crystals that formed resembled ice crystals. Sorry to hear about your samples. I've just been storing mine at RT in PBST, and that's worked OK for me. I wouldn't suggest leaving them in clearing solution for extended periods--I've actually seen some of my samples disintegrate when I've left them in CS for months and months...

Clara

balsorjl said:

Can anyone recommend a better tissue storage technique for after clearing than PBS or PBS-T at room temp? I find the sample continues to swell and the hydrogel collapses when left in this solution for too long.

Hi, I'm having trouble storing my samples in PBST at 4°C because I also noticed the hidrogel collapses after a few months. I remembered seeing in Deisseroth lab samples more than a year old in PBST and there were fine.
Any ideas of what can be happening to my samples? I was thinking to add sodium azide to PBST buffer, in case there is some microorganism "eating" my gel.

Jake

One of my friends recommend as follows:

Before storage, you have to remove residual SDS from the cleared tissues by incubating the tissues in PBS or PBST for 1-2 days at room temperature. Without this step, residual SDS will form precipitates in the tissue.

The cleared tissues can be stored in PBS or PBST at 4 °C for upto 6 months. To prevent microbial contamination, you may add sodium azide.

Clara

Thanks Jake, I'll try with sodium azide