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!