Unelectrophoresed, but clear...

GCJohnson

We randomly checked on this hydrogel perfused/embedded right hemisphere of a rat cortex that was sitting in clearing solution in a 37 degree incubator for the last 5 weeks. We just had not gotten to doing ETC on it but it looks by far better than any of the brains that we have put through ETC, and with next to zero tissue damage. We had not changed the clearing solution in more than a month. We are planning on trying to duplicate ASAP and see if changing the solution more often has any positive effect. We are thinking this might be an option that, if producing mass amounts of tissue, may end up being faster considering that your only limitation on brains going at once is incubator space.


https://docs.google.com/file/d/0B9rk7EERI_okX3ZzUlc5VHBBTVk/edit

nparker327

@GCJohnson Did that sample go into FocusClear before imaging?

nicolasrenier

That's great! Try to put you sample in Focusclear to see if you can get a total transparency. I was doing the same experiment on my own. It's been for 5 days at 37°c, it's not there yet, but I can see an improvement from the beginning!

I'm convinced that for samples smaller than whole brains, it is the right way to go!

nicolasrenier

Just on the side, I notice you have a dust/dead pixels on your iphone 5 sensor... You can get a new one for free from Apple, just show up in an apple store ! (I had the same problem). Sorry for this remark on the side.

Kristin_Engberg

Yes, tissue samples will clear in the clearing buffer over time as the SDS passively diffuses in and out of the sample. ETC is simply a method to speed this process up by promoting active diffusion of SDS. Passive diffusion from incubating the solution in SDS works too though if you have the time to do it. In fact, smaller samples (like 1 mm thick slices) should clear within a week via passive diffusion.

GCJohnson

Has not gone into focus clear yet but will report on how that turns out. Does anyone have a thought on what the limiting factor of clearing in this situation is? If we were to change the clearing solution more often (or even continuously in an immersion circulator) might that increase the rate of clearing? Again the point being that we are simply ameliorating all burning/melting possibilities by not doing ETC.

And thanks nic!

Fabio

Its diffusion of the SDS into the tissue,I would also try gentle rotation of the sample. If you have your tissue in a big volume of clearing solution I would not change it so often.

LauraLynch

I've got one hemisphere of mouse cerebellum in one 50 ml tube and the rest of the hemisphere in another. They're sitting in a 37 degree water bath. I change the buffer every morning. After one week, the tissue looks the way it would if I'd been doing electrophoresis for a day or two. I think I'm going to make this a practice for all the brains in line for electrophoresis. A little pre-clearing can't hurt, can it?

nicolasrenier

OK, so here is what I've got for the cortices :

https://docs.google.com/file/d/0Byy2Hiz1XfrdYVZsVE1rS0xsRzQ/edit?usp=sharing

You can see that 1 week of passive clearing is almost as good for transparency as 2 days of gentle electrophoresis.

However, I just did a DAPI staining, and the staining is very fuzzy and I can't image below 100µm because of background noise creeping up, which is ridiculous. I'm wondering if achieving transparency is not a good readout of a successful clearing. Maybe further clearing (longer ETC) could improve on the background autofluorescence?

KeithSiew

I'm thinking of trying this non-ETC approach on mouse kidneys and possibly human aorta samples... I'll keep ye posted.

ClarityNL

I am currently performing non-ETC clearing on mouse testis, don't know if they'll clear up as nicely as other tissue, but worthy a try since it was the only tissue available at the time.
Ill keep you posted on the progress.

willadler

nicolasrenier said:

I'm wondering if achieving transparency is not a good readout of a successful clearing.

I think this is a really important possibility.

Do you think it might also be the case for FauxcusClear? That is, you might come up with a recipe that achieves transparency but in the process causes imaging problems. You might have addressed this elsewhere in the forum, but I'm not quite caught up here.

nicolasrenier

The mounting medium could indeed be a problem for imaging, but I get similar bad imaging with the real focusclear. Still working on that...

TuckerA

I did the same thing with mouse insulin-GFP pancreas, after hydro-gel incubation with saponin I passively cleared with SDS for 8 days @ 37. This is what it looked like before being transferred to the PBST
Day 1
https://drive.google.com/file/d/0B_sWgVl1p-E7STlZdjRZYUZhcWc/edit?usp=sharing
Day9
https://drive.google.com/file/d/0B_sWgVl1p-E7bnVURzJUeUNySTg/edit?usp=sharing

Though after 5 days in PBST it has become cloudy again.

https://drive.google.com/file/d/0B_sWgVl1p-E7V1BiaVB3YjRpQWM/edit?usp=sharing

Fabio

This is normal, tissue gets more opaque in PBST than in Cleating buffer.
You can truly see the clarification once you put your tissue in FocusClear (match the RI of the tissue)

Nice pics!