FocusClear Alternative

Does anyone have an working alternative to Focus Clear solution.
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  • VarfVarf Posts: 13
    I work with 50% glycerol - not too bad. Also tried 100 and 75% but it seems that 50-45% is better.
  • Hi Varf do you have some pictures to share (clearing and imaging)?
  • @Varf do you have any precipitates in the samples with 50-45% glycerol?
  • @Varf do your brain shrink back to original size with 45-50% glycerol?
  • VarfVarf Posts: 13
    Hi guyz, I worked with kidneys not with brains. What I found is that 50% glycerol gives you less autofluorescence compare to other dilutions. Also, it was already mentioned somewhere on this forum that for brains 40% glycerol works well.
    Never had precipitation or any changes in sample size...
    Please note that I have no experience with confocal microscopy! So far I only have access to a common light microscope with couple of UV filters.
  • LinusMGLinusMG Posts: 20
    edited February 2014
    I had two cleared brain samples with nice fluorescence in 80% glycerol. After two days the white precipitate appeared. Following @David comment in FocusClear discussion I have just put one of the samples with the precipitate in a 40ºC waterbath. The precipitate started to disappear.

    Could it be that glycerol's high viscosity is pushing inside some residues?

    I will start playing with less glycerol-concentrated mounting media and incubation temperatures and I will keep you posted.
  • LinusMGLinusMG Posts: 20
    edited March 2014
    As @Varf mentioned what worked best for RI matching in my samples has been 50% glycerol.
    I have also tried 80% and 90% glycerol and the sample looked a bit more opaque (anyway I am not sure if the effect is due only to the swelling of the sample).

    I have noticed that in order to get rid of the precipitate it is good to incubate the sample at high temperature in 50% glycerol for some time.
    After one day incubating at 45ºC in 80% glycerol, when cooling the sample at room temperature some of the precipitate appeared again, after two more days incubating the precipitate disappeared completely even after cooling. 50% glycerol swells the tissue and the diffusion of the precipitate is faster.

    I also have tried to incubate a new sample in ScaleU2. Now it has been incubating for one day and I found that the fluorescence is preserved (in opposition to what I found with the first sample I cleared).

    I post some pictures:
    https://drive.google.com/folderview?id=0B9SnslqET-pLRWJiNzVzWm1jeTQ&usp=sharing
    The experimental sequence was: 1st 80%glyc with precipitate 2nd 80%glyc without precipitate and 3rd 50%glyc. I have noticed that coming back to 80%glyc after incubation at 45ºC in 50%glyc has better quality than the second picture.
  • So are glycerol and ScaleU2 the only viable options?
    Was nobody able to improve the FauxcusClear solution someone shared last year?
  • MRGneuroMRGneuro Posts: 5
    edited July 2014
    Has anyone tried the SeeDB solution for clearing? Their paper showed better results than ScaleU2 and doesn't cause the tissue to increase in volume.
    http://www.nature.com/neuro/journal/v16/n8/full/nn.3447.html
  • DUJDUJ Posts: 11
    I`m clearing kidney tissue and tried both Glycerol and SeeDB. I found SeeDB to give much better results in terms of transparency. The downside is the high viscosity, which makes it a bit hard to work with and you have to be careful not to introduce bubbles.
  • DUJDUJ Posts: 11
    Here are some images of the clearing process. First image is before clearing, the middle one after CLARITY lipid removal (in PBS) and the last image shows the whole kidney when mounted in SeeDB.

    https://drive.google.com/folderview?id=0B6kVu4hx1IA7aHFpaFlUQUxhbTA&usp=sharing
  • cheungmcheungm Posts: 1
    @DUJ‌ have you imaged any of your tissue? I'm curious if there's any difference in how it looks under the scope with SeeDB vs. FocusClear.
  • DUJDUJ Posts: 11
    I uploaded some confocal microscopy images in the folder taken with a 10X air objective at different depths. It is kidney tissue stained with anti-E-Cadherin antibodies.
  • ViktoriaBViktoriaB Posts: 1
    @DUJ‌ : Nice clearing result! What ETC parameters did you use? (V, A, temperature, time) Thanks in advance!
  • I
    DUJ said:

    Here are some images of the clearing process. First image is before clearing, the middle one after CLARITY lipid removal (in PBS) and the last image shows the whole kidney when mounted in SeeDB.

    https://drive.google.com/folderview?id=0B6kVu4hx1IA7aHFpaFlUQUxhbTA&usp=sharing

    So @DUJ‌, did you Clear with Clearing solution, and then mount with SeeDB?

    If so, did you directly put it in the seeDB (~80.2% Fructose), or did you pass it through a concentration gradient before mounting?
  • Have you tried RapiClear? It seems to have done a pretty good job for us but haven't tried FocusClear for comparison.
  • AdamAdam Posts: 31
    I had a look at comparing FocusClear and RapiClear 1.47 in 1mm brain slices. No obvious differences, but nothing quantitative.
  • VarfVarf Posts: 13
    Hi everyone. Have you seen Cell paper with the total mouse transparent? They proposed the Focusclear alternative there http://www.cell.com/action/showExperimentalProcedures?pii=S0092-8674(14)00931-3
  • DUJDUJ Posts: 11
    Sorry for a late response, been on vacation. @ViktoriaB For the whole cleared kidney I first cleared passively for 5 days, followed by 7 days of ETC at 50 degrees and 15 V. However, I now dropped the ETC step since it seems to have an impact on some proteins (Antibody staining showed no signal), and I now focus on clearing cubes of tissue (~1mm) passively.
    @EmileHashem I did the intermediate steps with a final conc. of 80.2% for the whole kidney. For smaller samples you do not need the intermediate steps and a lower final concentration can also be used to make the medium easier to work with (80.2% is very viscous), though this requires the samples to be fairly transparent already after the lipid removal step.
  • SuSamSuSam Posts: 6
    I don’t know why, but RIMS prone to crystallize at the surface of the liquid, and it seems that the hydrogel “dissolved” in RIMS and becomes sticky in the following processes. As an alternative, I tried both Focus Clear and RapiClear, and I prefer RapiClear for it's cheaper and showed no difference in clearing effects and the resulting image quality.
  • dgrobertsdgroberts Posts: 16
    The crystallization/sticky issue happens when RIMS isn't in a closed environment. I'd recommend trying a different mounting technique that completely encloses your sample. I'm familiar with your issue and I know it can be a pain, but RIMS has worked really well for us after resolving this.
  • SuSamSuSam Posts: 6
    Thanks dgroberts for your suggestion.
  • IreneIrene Posts: 9
    Hi, i've recently published an article on a new clearing agent (TDE) suitable with CLARITY that replaces Focus Clear. It is open access, the link is http://www.nature.com/srep/2015/150507/srep09808/full/srep09808.html
    Please ask me if you have any questions about it, it is very easy to do and very cheap. Furthermore it is suitable with different type of microscopy.
  • There is a new product on the market called EasyIndex. It is said to be less expensive than FocusClear but has much the same function.
  • Where can we find that product (EasyIndex)?
  • You can find this at Live Cell Instrument - www.chamlide.com
  • JakeJake Posts: 10
    I also found a commercial alternative. Here is a comparison of that with FocusClear: http://logosbio.com/download/pdf/The_X-CLARITY_Mounting_Solution.pdf. It is also less expensive than FocusClear.
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