CLARITY of Embryos

Jalal

I'll be posting images of cleared mouse embryos as they come. At the moment, I have tried 2 days at 30V with 37C buffer temperature. I think it's beginning to clear a little. Here is my first pass, uncleared on the left, cleared on the right:

https://docs.google.com/file/d/0B1fiJCzjdQ3qWFRiVTgzc3V6cmM/edit?usp=sharing

Thanks to @nicholasrenier and others for their comments and feedback.

Anyone else make progress with embryo imaging?

karlkoeh

@Jalal I am about as far as you are with some E9.5 and E13.5 embryos. I don't think the E9.5 will need ETC, so I am doing a week of soaking in the clearing solution. My E13.5 embryos look similar to yours after 48 hours at 20V and 40ºC. This might be as good as it gets until we add the focusclear. I will be performing immunostaining of the next 2 weeks. I'll post pictures once I've placed them in FocusClear.

I noticed you have some hydrogel dangling from the cleared embryo. I stumbled upon a great way to remove the last bit of hydrogel from the embryo surface: roll the embryos across the surface of a laid out kimwipe. As the hydrogel dries it begins to stick to the kimwipe and is pulled off the surface of the embryo.

Jalal

Thanks @karlkoeh for the suggestion. I do have hydrogel remaining and I will try your suggestion. The other problem I have is that I seem to have cooked my embryos to the point where the hydrogel is opaque on the periphery-- that's why the hydrogel was so visible only on the embryo post ETC, although both samples are submerged in buffer.

Here is day 4 after ETC 37C, 20-30V (no ETC on the left):
https://docs.google.com/file/d/0B1fiJCzjdQ3qVXdqcTFOODlvb1U/edit?usp=sharing

No much improvement.

While I haven't tried a brain to test for the effectiveness of my setup, it seems that embryos cannot be cleared to the same degree with CLARITY. I am considering a 2 week run next with low voltage.

nicolasrenier

Hi guys

I'm also interested in clearing embryos, but I wouldn't go with clarity. Embryos are much easier to clear than adult tissues, and can be more easily, cheaply, faster and efficiently cleared with other protocols. I recommend to try SeeDB, ClearT or 3DISCO for embryos over Clarity. I'm trying to compare those methods now to see which works best.

Jalal

Thanks for checking in @nicolasrenier. I would be interested to test those protocols as well. I went with clarity on principle because I felt that the approach may preserve antigens-- cross linking proteins to acrylamide. I would like to stain for certain surface proteins at some point. Also, I think 3DISCO or SeeDB require a few weeks to work and at that point I think proteins are decomposing. All speculation at this point.

However, it turned out to be much more complicated than I envisioned. I'm hoping a 2 wk clearing process at low voltage will do the trick. I think my setup is sufficient.

nicolasrenier

3DISCO seems to work really well for immunostainings (not so much for endogenous fluo). With an object as small as an embryo (I'm talking E11 to E14), just 2 days are enough with any of these methods to get a decent clearing. I'll report back when I have a good side-by-side comparison!

jbush

Hi @nicolasrenier, do you find that antibody penetration is good with the 3DISCO method?- do you have a method to improve ab penetration? I have tried SeeDB, Sca\e and ClearT on E11.5, E12.5 embryo heads, and clearing is ok with these, but I am now realizing that ab penetration is not great at these stages (which will obviously not be improved by these methods). I was hoping that Clarity would be the solution, but so far the Clarity protocol completely bloats and deforms the tissue (good morphology is another prerequisite for this whole thing). Does anyone have any thoughts on this?

karlkoeh

Hi @jbush, I share a similar opinion. The most exciting "potential" advantages of using CLARITY to stain embryos and smaller tissue samples are improved antibody penetration and being able to perform several rounds of immunostaining. ETC may be irrelevant.

Has anyone here been testing the CLARITY immunostaining protocol with 1 M sodium borate buffer? I went with room temperature instead of 37ºC because I didn't think the high temp would be necessary with E9.5 embryos. Unfortunately, a thick covering of crystals developed on the outer surface of the embryos about halfway through the primary incubation. The crystals dissolve in PBST (I did a quick test), but I have no idea if this will interfere with the staining process. Thoughts? Similar issues?

karlkoeh

Problem solved. 37ºC = no crystals.

Apparently, 37ºC is absolutely necessary when using this buffer.

nicolasrenier

@jbush Antibody penetration should be good enough for the stages you're studying. Have you tried using PBS-1% triton? In the seeDB paper, they do the blocking steps with 10% BSA, 1% triton in PBS. I've seen whole E18 embryos efficiently stained that way (the clearing was done with BABB).

I did a first test today with ClearT, and could "only" go at about 300µm inside the sample with a confocal. I still have to compare that with 3DISCO and SeeDB.

jbush

Hi @nicolasrenier, Thanks for the suggestion, i will try this. Currently blocking in .1%Triton, so 1% might improve things. By the way, I found SeeDB to be better clearing than ClearT (though slower) and although I haven't tried it, I understand that 3DISCO decreases fluorescence intensity signficantly. Sca\e works well, but its nearly impossible for us to orient the embryo in this solution, and we cant embed in agarose from there.

Jalal

Hi @nicolasrenier. I will try 3DISCO for embryos. Have you had success with this? Thanks for your input

nicolasrenier

Hi Jalal. 3Disco works great for me, following whole-mounts immunostainings. I can go as deep as the objective working distance allows me to (3mm on our setup) without any loss of signal.

Jalal

Hi @nicolasrenier. I can confirm as of today that 3DISCO works great for me as well! However, I had heavy bleaching of my GFP signal (I use a transgenic mouse expressing GFP under Nestin promotor). Could you give some comments on your approach? perhaps PFA fixation time and how long after DBE do you image?

As for ClearT2-- I did not observe any improvement in clearance..

SeeDB is TBD

Thanks for your advice early on, it really helped and I'm excited to see if I can get some good data now!

nicolasrenier

Jalal, 3DISCO is bad for fluorescent proteins in general, it quenches the fluorescence very fast. Clarity does a better job at preserving the fluorescence of proteins. I use 3DISCO exclusively with alexa dyes (immunostainings or tracings). You could do an immunostaining against GFP with an Alexa488 secondary, and then clear with 3DISCO, that should give you great results.

Also, I had poor clearings with ClearT, and can't get seeDB to work. I would be interested to see if anyone had a good experience with seeDB on embryos.