Clearing of postmortem human brain tissue

Anke

I was wondering if there are users on this forum that try to clear human postmortem brain tissue. In the paper it is stated that previous formalin-stored material (more than 6 yrs in formalin) was still suitable for CLARITY. I used Saponin in the hydrogel and tried passive clearing for a month on long-fixed (>1 yr) and short fixed (72hrs) material and there seems to be a large difference in clearing of the samples, where no clearing has been observed in 200 um long-fixed material compared to (beginning of) clearing of 3 mm chunck of short-fixed material.

Does anybody have similar experiences? Would it be advisable to switch to ETC for long-fixed material?

Thanks!
Anke

mhurry

Hi Anke, I was just wondering how you got on with doing CLARITY on human postmortem brain tissue? Did you set up the ETC in the end?

Thanks!

Anke

Hi!
I only tried passive clearing. One of my concerns was that the tissue expanded so much, which was not useful for our experiments. We ended up using SeeDB on thinner sections, as it does not disturb the morphology as much. What are your results so far?

mhurry

I see! Was tissue expansion the only reason for you switching to SeeDB?

We are just setting up CLARITY in our lab and I'm going to have a go with ETC as well as passive on various human brain sections. I'm about to begin some ChAT staining of passively cleared sections & will keep you posted. Any words of wisdom you have for processing human brain samples would be greatly appreciated too!

Anke

I tried several thicknesses: cortical blocks from 1 cm to 3 mm. Blocks with a lot of white matter are hard to clear, possibly because the hydrogel does not penetrate that well. I used unfixed tissue for this experiment.

The expanding was one of the reasons, the other one the costs. In my campus, several other people were having problems with the staining in mouse brain, which also did not give me a lot of hope. i would go with thinner sections to clear and a good antibody!

Manoj

Hi Friends,

I would like to try some human tissue samples (pieces of frozen and floating human brains tissue and 2-4 mm thick slices) for CLARITY or PACT/PARS.

Does anybody have protocols for the procedure tested for human samples. if you have protocol for it, please send me a text on
jaiswal.manoj@gmail.com

Thank You,
Manoj

Irene

Hi @Manoj I'm working on human brain tissue. You can find the protocol that i'm using for staining on: http://www.nature.com/srep/2015/150507/srep09808/full/srep09808.html