vDISCO Troubleshooting

[pineapple]

Hi!

I am trying to reproduce the vDISCO protocol and have been running into some issues when imaging with the lightsheet.

In the future, I hope to use this protocol to image fine nerve fibers in the face so having a quality clearing is very important to me.

I have been trying vDISCO using a PI staining (Cai 2023, Nature) to evaluate the quality of my clearing. The sample seems fairly clear however I am getting poor resolution in the brain.

The perfusion was good, the decolorization and decalcification steps seemed to be fine (heme eluted from the spleen and bones become flexible). the clearing I felt was followed to a tea.

Any advice based off these images?

[zacsimile]

Hi @pineapple. It’s difficult to tell what’s going on here, but it looks like the clearing may not have worked as expected. The blurring in the images you posted indicates that there could be an RI mismatch between immersion and sample. We ran into a similar problem using a simpler iDISCO protocol on kidneys. In our case, immersing the sample in DCM until it sank to the bottom of the tube resolved the issue. I see DCM is also part of vDISCO, though I’m not sure if this approach would help here. We worked it out by contacting the protocol’s developers. It might be worth emailing the vDISCO corresponding author to see what they suggest.