My brain is White!

JAGS

After 24h in the storage solution (PBST (0.1% Triton X) + Azide Sodium) my sample is white. I think that it's due to the detergent's precipitate. Could you help me? Had it happened to you anytime?

Adam

Is it white, or just cloudy? Cloudy is fine, it's just a refractive index mismatch. It should clear when put in focusclear/glycerol.

JAGS

I think that it's white, but it probably would be normal. You can see it in this URL: https://www.dropbox.com/sh/g4n1zb59lq3kjha/AAAeb6VarpMrVQCHQcSIpK8Pa?dl=0 .
What do you think about?

Fabio

Did you wash long enough in PBST? Could be residues from the SDS that precipitate at temp lower 15 °C
also your buffer is really cloudy... shouldt be

JAGS

It's possible, I left it 15ºC, and now it's fine.

https://www.dropbox.com/lightbox/home/CLARITY

Thank you Fabio!

hydrazine

My sample is white when in PBST also.

JAGS

Have you left it to15°C or RT?

hydrazine

It has been at room temp. I have not incubated it in a refractive index matching solution yet, but I suspect it will return to being clear.

dgroberts

For me, even washing in PBST at 40°C leaves a white ppt that never fully corrects the refractive index. I've noticed that the quicker I wash (shorter duration with many solution changes), the clearer my samples are after matching in FocusCLear or RIMS, and the greater my image depth without signal drop-off is.

dgroberts

Does anyone else have any other recommendations for eliminating the white ppt problem? I've particularly noticed its irreversibility after being stored at cooler temperatures. I've had some success putting these samples back in clearing solution on the heat shaker for ~1 day, and then washing again in PBST before refractive index matching, but I can't quite get them as translucent as some of my other samples, and consequently only get 1-2mm of good penetration on confocal (as opposed to 3-4 mm with a sample washed quickly after clearing and stored at room temp. in its RI matching sol'n).

yizhong3x

How about using the 0.9％ nacl？I haven’t tried it.

RLaws

Ive had the same problem with this but ive managed to get good clearance on a 1-2mm thick mouse brain sample by performing the initial wash at 37°C. If you let the SDS precipitate once the effect is fairly permanent. If your looking at a whole brain you could try perfusion with 37°C PBST to make sure all the SDS is cleared but i dont know if that will work after the hydrogel and clearing steps.

dgroberts

@RLaws I also get good results with 2mm mice brain sections, but I'm currently imaging whole brain hemispheres and these have been difficult to image cleanly all the way through. I get signal drop-off starting somewhere between 2-4mm and it depends entirely on how well I avoided precipitation, which is to say how well I washed after clearing. I have have ran many many experiments to prevent it, and so far the best technique is to actually wash for less duration. It seems as though any residual SDS inside my samples diffuses out well when subjected to RIMS, but not so much in PBST. This kind of leads me to believe that it might be the salts in PBST that are precipitating in this case and not SDS.