ETC clearing end point

nicolasrenier

This is just a quick question I wanted to ask : how can we know when the ETC clearing is complete ? How the tissue (brain) should look at the end of this step? More specifically :

- In the first runs I did, I always end up with a yellowish tissue. Is this normal, or I am doing something wrong (Temp too high? Runs too long?). All the people I talked to have the same observation.

- Is the brain supposed to be transparent at this step? My brains are twice bigger after the ETC clearing, and are a light permissive, but definitely not "see through".

Kristin_Engberg

We don't yet know of a foolproof way to determine when clearing is finished, but when in doubt, I would leave the tissue in the ETC chamber an extra day or two. The brain will most likely not be completely transparent immediately after ETC clearing. It does remain yellowish in color. Swelling during the ETC clearing is also expected. Once the tissue is transferred to the FocusClear solution, it should return to it's normal size and become transparent.

gjones

The change in colour could also be due to the voltage being too high, burning the tissue. We had our ETC chamber manufactured and as such didn't follow the directions provided, only the dimensions. The result was that our electric field strength at 60V is higher than that of the ETC chamber as described in the chamber construction protocol, meaning that 60V was actually too high. From now on we use a much lower voltage (~30V) with which we achieve significant clearing in the same amount of time as 60V would have. Your yellowish colour could perhaps be due to the same reason ours was. Just a suggestion!

mk1

Does anybody have a picture of what the brain should look like after the ETC, but before clearing with FocusClear?

joelrosiene

https://docs.google.com/file/d/0B6RE82086JGFQW13TGNBTVVtZG8/edit?usp=sharing
https://docs.google.com/file/d/0B6RE82086JGFN0lJUzE4X1djR2s/edit?usp=sharing
https://docs.google.com/file/d/0B6RE82086JGFeXJmSlZzVmVZTnc/edit?usp=sharing

Here you go @mk1. That brain is a little yellowed though, I am re-running at lower voltage just now.

mk1

Thanks! How long (at at what voltage) was that sample run?

lijun

@joelrosiene.Your brain is fairly transparent after ETC.I want to know if you did the transcardial perfusion before tissue embedding.

joelrosiene

That brain was run at 50 volts for 2 days, although I do have a nonstandard chamber design, so my voltages will differ slightly from yours.

I did do the perfusion. It seems like a very important step.

rspence

hello everyone, wanted to share my experiences with clarity because i need a lil help. so far i have recreated the chamber and methods from the nature paper. i get to about day 4-5 and thats when the brains stop clearing and stay a kind of yellow opaque color. https://docs.google.com/document/d/1LCdJuVoDXoUhkCIXXR3eOEJRtkDBk1ncrh1FGjfyUfc
im running at 40V 40 degrees. any ideas? i was thinking about changing the solution after 3-4 days due to some mention of how critical the pH is. also, just a random question but has anyone yet been able to obtain images from microscopy from a cleared brain similar to what was in the paper?

agal1

@joelrosiene, what temp was you 50V, 2 day run at?

Thanks.

karlkoeh

@rspence, that looks like fairly decent clearing straight out of the chamber. The issue most people seem to be having (including me) is that the brain does not entirely clear until after it is placed in FocusClear. The issue is that FocusClear is extremely expensive and difficult to obtain. Refer to FocusClear discussion for more on this issue. Also note that in the original article the authors cleverly use black and white images of the fully cleared brain. Thus, some yellow tint to the tissue is probably normal!

rspence

@karlkoeh, thanks. i have 5ml of focus clear so i will check the discussion and give it a go. will let you all know what results i get.

stuberlab

Here is a pic from our first etc run pre glycerol; 3 days at 25 V (~0.8 A) and 40 C.

https://docs.google.com/file/d/0B0pHbI3Zgu3gYmpoZmx6UkFYNzg/preview

I have it in 85% glycerol now in room temp. I noticed the swelling dampened, but the yellowish tint still surrounds the outer edges. I assume that is due to overcooking and not having any focus clear, so I guess I will try even lower voltages. Has anyone seen the yellow tint disappear after glycerol washes?

joelrosiene

@stuberlab I have not found 85% glycerol to be particularly effective at RI matching, but perhaps you will find differently! I would be very interested to hear of your success. Right now most of my RI matching is being done in a DMSO solvated diatrizoic acid solution (saturated). The only issue that I have is the production of bubbles within the tissue.

joelrosiene

Sorry for not addressing you @agal1, My temperature for my most successful runs (and the one you cite) was 37C. Keep in mind, when planning your own run based off my successful result, that I have a much longer distance between my electrodes, which should mean that I have a chamber with higher resistance than the standard design.

stuberlab

@joelrosiene thanks! Does the yellowish tint affect your imaging of fluorescence? I haven't tried imaging it.

joelrosiene

To be honest I have great difficulty with fluorescence. The first brain that I successfully cleared was a Thy-1 GFP in an attempt to replicate the results of the original paper, but I am seeing a lot of autofluorescence, and my RI matching media resulted in bubble formation within the tissue for the first sample I tried. In a good RI match, the tissue should be relatively clear, and the yellowish tint should diminish.

https://docs.google.com/file/d/0B6RE82086JGFdzFWd0ktTnREVGM/edit?usp=sharing
https://docs.google.com/file/d/0B6RE82086JGFQ2cyMi1rcnhuZUU/edit?usp=sharing
https://docs.google.com/file/d/0B6RE82086JGFVmgya3Q1SGVjLWc/edit?usp=sharing
https://docs.google.com/file/d/0B6RE82086JGFUDlRUmdGMmg4dE0/edit?usp=sharing
https://docs.google.com/file/d/0B6RE82086JGFMmJBR2RqT0o3dUU/edit?usp=sharing

See these pictures for my recent experiments with RI matching. Those are all in the DMSO solution. (quartered Thy-1 GFP)

lijun

@joelrosiene. what is method you uesd to seal the imaging chamber? when I seal the chamer use the Blu Tack or PDMS,bubbles always be introduced into the chamber ,and the Blu Tack will lose the sealing capacity when contacting with water or FocusClear.

stuberlab

@joelrosiene those pics look good. For long term storage, do you store the tissue in the RI solution or pbs in room temp?

joelrosiene

To be fair, I took those pictures before sealing the chambers down. Maneuvering the chambers around to try to seal them almost always resulted in bubbles, which look bad

I used clear nail polish to seal them - I have the Quik-Sil stuff they used in the paper, but I deemed it a bit too expensive to use on these early trials. that said, they didn't seal perfectly, it was very very difficult to get a seal. I may have spent 2 or 3 hours trying to seal 4 slides perfectly with no bubbles.

I was planning to stores the tissue in the RI solution, because of the way I was planning to seal them.

Andy_Donaldson

@joelrosiene , what size slides did you use to seal your samples in with the Blu Tack? Are you using Scale A2 as your RI medium?

joelrosiene

@Andy_Donaldson I used boring old 3"x1"x1.0mm Fisher slides. I'm using the saturated DMSO/diatrizoic acid solution mentioned above, mostly because I was worried about ScaleA2 or A4 quenching fluorescence.

stuberlab

@joelrosiene I just order the stuff to make focus clear. What was your concentration for your dmso solution? Thanks!

joelrosiene

@stuberlab I am using a saturated solution, which seems to be about 10-12% wt/volume. I heat the DMSO in a water bath to 50C, and vortex to get the diatrizoic acid into solution. I then add 0.5 molar equivalent of NaOH to balance out pH (even though there is no water), and then I add Tween20 to 0.1%, per Nic Renier's suggestion.

LauraLynch

Back in March my PI came up with a recipe for what we're calling "FauxcusClear." We tested it against the real stuff using 2-photon laser microscopy and saw no difference. Ours is a qualified success: we've had other problems that are most likely due to previous steps (autofluorescence, loss of true fluorescence) so I can't say whether or not our version works for sure. But if you want to try it, below is our recipe. Please help me make it better if you see anything that we're doing wrong:

We make a 70 mM stock of NADPH in 0.01N NaOH and add 1.425 ul in 100 ml final volume DMSO. We also add 0.27 mM EDTA (from a 500 mM pH8 stock I had for other uses), 1% Tween 20, and 12% w/v each of diatrizoic acid and sodium diatrizoate. Then we sonicate the solution until everything is dissolved. I've been storing the solution in 20 ml aliquots in scintillation vials (one brain/vial) in the dark at room temperature. We've already shared this recipe with @nicolasrenier and @nparker327. Maybe you guys can chime in?

nicolasrenier

Hi @LauraLynch, I'm just moving the discussion to the Focusclear topic ! See you there...