Post pictures of cleared tissue

Suzschindler

Please post some pictures of your cleared brains, including the conditions under which you ran the ETC (20 V x 7 days).

LauraLynch

OK, here's a whole pile of pictures. I took timeline photos with my phone; the quality isn't great, but you can still see the gradual color changes.

These are my best two brains so far. The rest were either fried beyond photography or just too fried. The electrophoresed sample was mishandled before clearing (it was overfixed), so, while we had the least amount of autofluorescence to date, and the structure was beautifully preserved, we still had autofluorescence and our GFP signal was gone. We have not yet looked at the passively cleared tissue.

Enjoy and/or help!

https://drive.google.com/folderview?id=0B5H6exz8a5XAcXZ1YXRBaXFsNWM&usp=sharing

Suzschindler

Thanks, that's helpful. I was most interested in the passively cleared sample. What were the conditions for this? 37C in a 50 ml tube for 8 weeks?

Also, I noticed your cell strainer was parallel to the bottom of the chamber. When you ran the sample was the cell strainer perpendicular, to decrease the distance between the electrodes?

LauraLynch

I've since changed the configuration so that the cell strainer is vertical. I cut the plastic edge off of a second strainer and fitted it into the first so that it makes a container.

The passively cleared sample was in a 50 ml tube, in clearing buffer, at 37 degrees. The buffer was changed daily for 21 days (three weeks).

We'll be doing microscopy on that sample within the next few days.

Jalal

Looks great Laura, thanks for posting. I'll also try passive clear on some samples and will post the results. Please do update on the clarity of the images with this approach when you get a chance.

hou

electrophoresed Tdtomato mouse brain with 450 mA at about 37 degree for 4 days
http://pan.baidu.com/share/link?shareid=3605270445&uk=3140964649

jmeeks

@hou: this is one of the better samples I've seen. Thanks for posting. Especially impressive was the illustration of the partial obliteration of the tdtomato signal (pinkness). Was this sample in CLARITY clearing solution at the time you took the picture?

We have been able to partially clear several samples, and were able to stop ETC step when our tissues showed similar partial loss of tdtomato. We think we understand what is happening, and are taking steps to test our hypothesis. I hate to tease, but I don't want to get anyone's hopes up without proof that we know how to avoid the destruction of tdtomato.

Fabio

Well got time to take a quick picture. Here is a cleared brain. The brain is floating in the solution, so the distance to the text is maybe to big. Still is a good clearing.
https://drive.google.com/folderview?id=0B4CxwuUeXqnOWk9mdm9QLXA3bmc&usp=sharing

patiprat

Hi Fabio nice pictures of the cleared tissue. Could you please share the cnditions you used for ETC clearing ? Thank you.

Fabio

Performed in a standard ETC camber. The ETC settings were 20 V, 37°C, 190 h. Buffers as in protocol. The washed in PBST and mounted in FocusClear

lijun

@Fabio hi,did the brain you've imaged be incubated in FocusClear ?or just take the picture when the brain was still immersed in the SDS clearing solution ?

Fabio

The posted pictures, the brain is in FocusClear

rspence

@Fabio and others, thanks for all your comments on this forum, they are very helpful for me. one more question for you, did you find that focus clear helped to further "clear" your sample, or is all of your clearing done with ETC, thank you.

Fabio

the transparency comes from the focusclear

aysegul

Thank you so Fabio !

Shashank20

Hi Everyone,

In parallel to ETC-mediated clearing I was also trying to passive clearing of whole mouse brain in SDS buffer. Passively cleared brain is now transparent. My query is if one gets rid of all the lipids from brain while passively clearing the brain when it looks transparent or additionally one has to run an ETC for few days to be sure of lipid removal ? Thank-you

willadler

@LauraLynch

That video looks great. Have you tried to image any passively cleared tissue yet?

hydrazine

This was taken after about 1 month of passive clearing.

http://imgur.com/sJtYtUy