Hi Michael,

I had been exposed only to BABB-based clearing technique. I am just carious, is your question triggered by shortcomings which you see in the aforementioned method or is it merely a general question? Hopefully, we can hear from other researchers who have tried the above alternatives.

The reason I ask is that I wonder whether other techniques would lead to less shrinkage and better "survival" of antibody labeling.. All mentioned techniques were used for confocal microscopy on sectioned brains, and worked relatively good there - but I wonder how well they'd work after embedding in agarose, and whether there were established (adapted) protocols around for OPT imaging.

I've attended the London CLARITY meeting and it seems like nobody got it working outside of the Stanford/MIT labs for brain imaging, so I currently want to investigate alternatives that could especially leads to less morphological alterations/shrinkage and preserves antibody labeling.

We've tried the ScaleA2 method in our lab:

http://www.nature.com/neuro/journal/v14/n11/full/nn.2928.html

However, this causes the volume to increase by double.

Thanks Aaron, we had similar problems with volume increases when using ScaleA2 in our institute for confocal imaging, which is why I excluded it already from my possible alternatives..

Michael, I have PMed you some contacts of OPT researchers who can probably answer your question.

Hi,

I am currently trialling the 3DISCO method on whole mouse brains. The clearing seems to work well and there is no obvious change in volume. We haven't got as far as imaging yet.

I'm hoping to find a way to immuno-label the tissue in order to carry our multiple stains, i.e. tag microglia and image the brain, then remove the immuno and relabel for GFAP for example.

Any advice is welcome!