Questions on CLARITY process (with pictures)

dbryantdbryant Posts: 6
Hello all,

I have recently started working with the CLARITY tissue clearing process. I have completed three rounds of clearing with a mouse liver (no images), a whole mouse brain, and a whole baby cuttlefish. I am currently working on a second whole mouse brain. I have the progression images as well as images of my ETC setup posted on a blog here: http://dmbryant14.wordpress.com/. Throughout this experience I have accumulated some questions that only people who are currently working with CLARITY can answer, so I have come to you!

1. Is the clearing solution supposed to appear cloudy? My clearing solution used to appear cloudy and have an initial pH of about 6.3, but I recently purchased a new bottle of SDS, and now my clearing solution appears transparent with an initial pH of about 5. I'm assuming the transparent version is better, because a transparent clearing solution makes more sense. I have images of both samples posted on the blog listed above.

2. Is the “E” configuration of the electrodes efficient for electrophoresis? Does most of the current occur between just the terminal points of the electrodes or uniformly throughout? I think that the current may not be uniform unless the electrodes are exactly the same distance apart in all places, but I don't think that the current is concentrated at the end of the electrode. I was asked this question and didn't have a solid answer, so I wanted to ask the community.

3. At this point in time I am sanding my electrodes (mainly the "negative" one) after each iteration of electrophoresis to clean them of black residue. It has been suggested that immersing the electrodes in a vinegar solution and running a current through them could possibly cleanse them in way that eliminates the damage that sanding causes. Do you think this method would work? How do you handle the cleaning of your electrodes?

4. I am currently using the 80% Glycerol for index matching, because I don't think the FocusClear is an economical option for my particular situation. If any of you have tried using just Glycerol, in your opinion, how much of a difference is there between 80% Glycerol and the FocusClear?

5. The protocol is a bit unspecific (mainly because of a lack of commas) on the conditions of gel polymerization. I can interpret the instructions to mean put the tube on a rotator, and then put the rotator in a 37C water bath, and then put the water bath in a 37C warm room. That really isn't an option for me so I currently put the tube on a rotator and then put the rotator in a 37C warm room. Also, since the tube is on the rotator, it takes about 5-7 hours for the gel to polymerize. If I just had the tube stationary I don't think it would take so long. I'm fairly confident that I am performing the dessication correctly. Your thoughts?

I think that is all my questions for now. On a side note regarding question number five; I think I found a good way to remove tissue from the gel. It worked for me during this most recent sample, and perhaps it may work for you. I used my gloved hands to remove the bulk of the gel. Once I was down to the tedious part of the removal I took some tweezers and perforated the gel as close to the tissue as possible. What I mean by this is that I clamped onto the gel near the tissue and then tried to pull the gel off. Trying to pull the gel off with the tweezers was rarely successful, but the perforation caused by the attempt made pulling the gel of with my fingers much easier. I have seen mentioned on this forum that using kimwipes is a good way to remove the remaining little bit of residue so that is what I do as well.

If you have any comments regarding my setup after looking at the pictures of it on the blog posted above, please post the comments on this forum and not on the comments of the blog. Thank you all for your time and consideration!


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    I just started with the protocol, but I might have a few answers.
    1. The clearing solution should be crystal clear, and you should start clearing your samples with a solution with a pH around 8.5. Just add NaOH till 8.5.
    5. I'm currently not degassing my samples in vaccum, I just take the sample of 4C to the water bath at 37C (stationary) and the gel polymerization is done in 2-3 hours.
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    FabioFabio Posts: 62
    I can quickly answer maybe your 1st question.
    I had also a batch of SDS that gave me a white clearing solution.
    I called the company and got a new one (Sigma)

    With the new batch the clearing solution was clear as always.

    So I would not risk using a blurry clearing solution. It muss be some kind of contamination in the SDS.
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    dbryantdbryant Posts: 6
    Thank you for your comments! It's good to know that I have (hopefully) better clearing solution now. @LeandroCTP I bring the pH to 8.5 before I clear, but I take a pH measurement before I add the NaOH just as an additional measure to make sure the solutions are consistent.
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    LinusMGLinusMG Posts: 20
    Your pictures remind me the results that I had using slow flow rate in the chamber.

    Now my best results are using 20L/min, 50ºC and 30V for 2 days and then 4-5 days of passive clearing.

    1.- I have not had any problems with the clearing solution, always clear.

    2.- For me as long as the two electrodes are symmetric the current will occur uniformly across the two planes they define. More lines would imply more uniform current across the plane.

    3.- I didn't clean the electrodes and I focused on isolating the sample from them (I also use a part of another sample holder for enclosing it).

    4.- I'm waiting for a very good sample before spending the FocusClear we bought. But for me 80%glyc is not too bad. A good clearing gives good enough images Soon I will post some pictures.

    5.- I use a 37C waterbath too without any problems (I degas anyway).
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    dbryantdbryant Posts: 6
    Thanks LinusMG! I look forward to seeing your images! I'll consider trying out those parameters.
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    jdscottjdscott Posts: 8
    Quick note on your 4th question - if you are interested in continuing with liver samples, you may want to image in 40% glycerol rather than 80%. Liver tends to have a lot of native background fluorescence, and this some people have noted (our lab included) that autofluorescence is more noticeable in 80%. We've never used Focus Clear but have been able to get nice images with glycerol.

    Echoing LeandroCTP, I also don't use a vacuum to degas - I just bubble nitrogen over the surface for a few minutes, seal, and place in 37C for about 3 hours (no rotation; works fine).
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    A couple more thoughts...

    1. I agree with others that it is probably best to use the newer batch of SDS which produces clear solution. Perhaps your first batch was contaminated?

    2. As stated above, the current should occur uniformly between the two electrodes. I always make sure that the electrodes are as symmetric as possible in the chamber to help with this. In other words, when you look at your chamber from the side, the electrodes should look superimposed on each other, like there is only one.

    3. We've found that physically rubbing off the black residue every 2-3 days of use using a microfiber cloth and tarnish remover is effective in cleaning the electrodes sufficiently such that the residue does not transfer to the surface of the tissue. An easier method that we now use is to simply reverse the polarity of the electrodes every 2-3 days and let ETC run (without the sample inside the chamber) for about 30 minutes before placing the sample back in the chamber and returning to the normal polarity set-up. This is really efficient at cleaning off the black deposits. We've found that it only works if you start with a brand new chamber (clean electrodes) and always clean them after 2-3 days of use. If the black residue builds up over time, physical rubbing seems to be the most effective method of cleaning.

    4. We've found FocusClear to be the best mounting solution when you want to image deep into the tissue. For your purposes, glycerol may be ok, you'd just have to try.

    5. As long as you get a gel in ~3 hours, whatever methods you're using are probably ok. Rotation or shaking during polymerization is nice, but not necessary. Placing the tube in a water bath that has already been equilibrated to 37C will help to bring the temperature of the solution up quicker than air, so I suggest putting your sample in a water bath in the warm room. Make sure your warm room temperature is steady, as we have occasionally had problems with gels not polymerizing due to the warm room temperature dipping down to 34 or 35C.
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    dbryantdbryant Posts: 6
    Thanks @jdscott and @Kristin_Engberg! I will definitely try reversing the polarity to clean the electrodes. @Kristin_Engberg I'm curious whether or not you have your electrodes permanently bound to your chamber due to your brand new chamber comment in #3. I have my electrodes attached with silicone sealant, so I am able to remove them completely to clean them and then reattach.
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    VortexamVortexam Posts: 8
    Did you use a dissected tissue for liver or you used a formalin embedded tissue?
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    @dbryant - we have the electrodes permanently bound to the chamber, so what I meant to say was that it works if you start with unused platinum.
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    dbryantdbryant Posts: 6
    @Vortexam We removed the liver from the mouse and then immediately placed it in the hydrogel monomer solution.
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    dbryantdbryant Posts: 6
    edited April 2014
    I have completed the clearing process and posted the pictures of the most recent mouse brain on a blog here: http://dmbryant14.wordpress.com/. It seems that the clearing works fine until it hits a certain point. I'm talking in particular about the image with sample held up to the light. There is a definite border between optacies. The picture with the brain on the cell strainer shows another feature that I thought was interesting. There appears to be some black lines that have formed. I think that if the brain was burned there would have been more of a black spot than black lines. I used ~25V for the clearing process. Overall, this brain is an improvement over the last clearing iteration. How does it compare to some of the brains that you all have produced, and what may be the cause of the difference?
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