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        <title>Hydrogel Embedding — CLARITY forum</title>
        <link>https://forum.claritytechniques.org/</link>
        <pubDate>Sat, 08 Aug 2026 01:33:39 +0000</pubDate>
        <language>en</language>
            <description>Hydrogel Embedding — CLARITY forum</description>
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    <item>
        <title>No polymeryzation</title>
        <link>https://forum.claritytechniques.org/discussion/212/no-polymeryzation</link>
        <pubDate>Tue, 26 Mar 2019 23:07:47 +0000</pubDate>
        <category>Hydrogel Embedding</category>
        <dc:creator>XiaoTong</dc:creator>
        <guid isPermaLink="false">212@/discussions</guid>
        <description><![CDATA[Hello All!
I am new to CLARITY work. I made my very first batch of hydrogel monomer solution two months ago (4% acrylamide, 0.25% Azo initiator, 1xPBS pH 7.4, and 4% PFA in ultrapure water) following the protocol precisely. The polymerization was great at my first attempt. Then after several weeks I did another embedding with the same batch of monomer solution. Surprisingly, after 3-hour 37 degree incubation, the solution is still almost in liquid form and no apparent polymerization. The hydrogel solution was stored in -20 degree and thawed on ice. I made a new batch, but still no polymerization. Any thoughts on how to solve this problem or trouble shoot further?
Thank you!]]>
        </description>
    </item>
    <item>
        <title>Hydrogel Solution Not Working</title>
        <link>https://forum.claritytechniques.org/discussion/207/hydrogel-solution-not-working</link>
        <pubDate>Mon, 04 Jun 2018 19:08:25 +0000</pubDate>
        <category>Hydrogel Embedding</category>
        <dc:creator>DRTMCL</dc:creator>
        <guid isPermaLink="false">207@/discussions</guid>
        <description><![CDATA[Hello All, 
My lab is relatively new to CLARITY work. About 6 months ago, we were able to get a solid batch of embedding to work, to the point that we were successfully embedding tissue and passively clearing them. We followed the CLARITY wiki's embedding and clearing directions to the T. The first time, we made all of our own hydrogel solutions and found this success. Then about 3 months ago we were no longer able to get the polymerization to occur. The initial batch just stopped working. Since then, we have tried remaking solutions, improving degassing, buying standard stocks, monitoring pH, using replacements (APS instead of thermal initiator, fresh BIS, fresh Acrylamide) to try and nail down what exactly was causing the polymerization to fail, only to be left scratching our heads and unable to replicate. Turning to you guys, do you have any recommendations? Has anyone encountered similar conditions? Any guesses at all will probably help us solve this puzzle. Thank you.]]>
        </description>
    </item>
    <item>
        <title>Easy removal of hydrogel?</title>
        <link>https://forum.claritytechniques.org/discussion/177/easy-removal-of-hydrogel</link>
        <pubDate>Wed, 17 Feb 2016 10:19:51 +0000</pubDate>
        <category>Hydrogel Embedding</category>
        <dc:creator>wwhannemann</dc:creator>
        <guid isPermaLink="false">177@/discussions</guid>
        <description><![CDATA[I am testing the CLARITY method using 500um 4T1 cells. However, am finding it very difficult to remove the excess hydrogel. This resultantly makes it difficult for the clearing solution to penetrate the sample. Does anyone have some recommendations?]]>
        </description>
    </item>
    <item>
        <title>No Polymerization...</title>
        <link>https://forum.claritytechniques.org/discussion/199/no-polymerization</link>
        <pubDate>Wed, 14 Jun 2017 19:33:21 +0000</pubDate>
        <category>Hydrogel Embedding</category>
        <dc:creator>EmRy2</dc:creator>
        <guid isPermaLink="false">199@/discussions</guid>
        <description><![CDATA[I have a hydrogel solution with acrylamide, bis-acrylamide (both from Bio-rad), Va-044 from WAKO, PBS, and H20.  I fixed my samples for 24 hrs in  4% PFA before placing them in the hydrogel solution for three days.  Instead of using a desiccator, I have tried both Peanut oil alone as well as Peanut oil after removing the air in a vacuum from the hydrogel.  However, both sets of gel have not polymerized after sitting in a 37 degree water bath for more than three hours.  I have tried increasing the acrylamide by 1%, and that has not worked either.  Any suggestions on where else I may have gone wrong?]]>
        </description>
    </item>
    <item>
        <title>No polymerization...at all.2 batches.</title>
        <link>https://forum.claritytechniques.org/discussion/178/no-polymerization-at-all-2-batches</link>
        <pubDate>Mon, 29 Feb 2016 14:48:01 +0000</pubDate>
        <category>Hydrogel Embedding</category>
        <dc:creator>cytokine</dc:creator>
        <guid isPermaLink="false">178@/discussions</guid>
        <description><![CDATA[I've read the prior posts re these problems. The only difference is that we're mixing our own acrylamide  (sigma a9099-100g) and bis (sigma m7279-100g) and making our own 16% pfa solution. The va044 is the wako one. 
Following protocol...Degas and nitrogen flush. Tight cap. Incubated at 37...and nothing. Made second batch changed pfa ph to 7.45 after reading posts recommending this. Nothing. 
Took first batch out  overnight from freezer and left at room temp in conicals planning to discard. No polymers. 
Left first and second batch in single conical at 38 water bath overnight. Nothing. 
So I wonder what next? Should we stick with commercial solutions. Are folks mixing their own? What should we look out for? Any advice is immensely appreciated. Thank you. ]]>
        </description>
    </item>
    <item>
        <title>Tissue Removal from Hydrogel</title>
        <link>https://forum.claritytechniques.org/discussion/35/tissue-removal-from-hydrogel</link>
        <pubDate>Thu, 25 Jul 2013 01:40:02 +0000</pubDate>
        <category>Hydrogel Embedding</category>
        <dc:creator>gjones</dc:creator>
        <guid isPermaLink="false">35@/discussions</guid>
        <description><![CDATA[Hi All,

We're having issues with removing the sample from the gel.
The gel is extremely rubbery and as a result there is often a significant amount of gel left behind on the sample.
Is anyone else experiencing this or could shed some light onto the reason as to why this would be occurring?

Thank you!]]>
        </description>
    </item>
    <item>
        <title>Whole-brain clearing in 8% SDS</title>
        <link>https://forum.claritytechniques.org/discussion/153/whole-brain-clearing-in-8-sds</link>
        <pubDate>Sat, 07 Mar 2015 20:38:43 +0000</pubDate>
        <category>Hydrogel Embedding</category>
        <dc:creator>BJ2014</dc:creator>
        <guid isPermaLink="false">153@/discussions</guid>
        <description><![CDATA[Has anyone tried clearing a whole mouse brain in 8% SDS after hydrogel embedding? I don't have the resources to do PARS clearing, so I am thinking of putting a perfused brain in hydrogel overnight @ 4degC, followed by 3-4hr incubation @ 37degC water bath. Any thoughts on the plausibility of this? ]]>
        </description>
    </item>
    <item>
        <title>Two alternatives to the nitrogen purge for the hydrogel</title>
        <link>https://forum.claritytechniques.org/discussion/118/two-alternatives-to-the-nitrogen-purge-for-the-hydrogel</link>
        <pubDate>Tue, 05 Aug 2014 19:46:44 +0000</pubDate>
        <category>Hydrogel Embedding</category>
        <dc:creator>Genesprite</dc:creator>
        <guid isPermaLink="false">118@/discussions</guid>
        <description><![CDATA[   I attended the CLARITY workshop,  and while there, Kristin mentioned that someone was using Peanut oil on top of the hydrogel solution, or filling the tube to the top, to prevent oxygen from inhibiting the polymerization. These sounded like good alternatives to the nitrogen purge. The main problem with filling a 50 ml conical tube to the top with hydrogel, is the waste of reagent. 
   The first thing I tried was transferring the brains to 15 ml conical tubes and filling them all the way up to the tippy-top before closing the tube and placing in a 37 degree water bath. This worked great!! It isn't too hard to remove the mouse brains from the 15 ml conical, and it uses less hydrogel. I did have to use a metal spatula to get it lose, but it was fine. I also recently tried adding sunflower oil to the top. I didn't have peanut oil, but I did have sunflower oil on hand, it also worked great! This saves the space and cost of nitrogen tanks, vacuum pumps and desiccator. Definitely a big thumbs up.]]>
        </description>
    </item>
    <item>
        <title>Is A4P0 w/ photoinitiator stable in 4degC fridge?</title>
        <link>https://forum.claritytechniques.org/discussion/152/is-a4p0-w-photoinitiator-stable-in-4degc-fridge</link>
        <pubDate>Thu, 05 Mar 2015 17:29:56 +0000</pubDate>
        <category>Hydrogel Embedding</category>
        <dc:creator>BJ2014</dc:creator>
        <guid isPermaLink="false">152@/discussions</guid>
        <description><![CDATA[I seem to have made a lot more A4P0 (with photoinitiator) than I need and would greatly appreciate any advice about storing it (if it can be stable). Thanks]]>
        </description>
    </item>
    <item>
        <title>Why a Hydrogel solution?</title>
        <link>https://forum.claritytechniques.org/discussion/151/why-a-hydrogel-solution</link>
        <pubDate>Fri, 20 Feb 2015 16:07:26 +0000</pubDate>
        <category>Hydrogel Embedding</category>
        <dc:creator>marine_dubois01</dc:creator>
        <guid isPermaLink="false">151@/discussions</guid>
        <description><![CDATA[Hey !

I have a question : Why we use a hydrogel solution and not a another gel ? ]]>
        </description>
    </item>
    <item>
        <title>Gel polymerization does not work</title>
        <link>https://forum.claritytechniques.org/discussion/31/gel-polymerization-does-not-work</link>
        <pubDate>Tue, 23 Jul 2013 12:54:58 +0000</pubDate>
        <category>Hydrogel Embedding</category>
        <dc:creator>jimichu</dc:creator>
        <guid isPermaLink="false">31@/discussions</guid>
        <description><![CDATA[Dear forum members.
I have been having problems with the HYDROGEL polymerization. Do you think it is necessary to fill up the vacuum chamber with nitrogen after degassing?. So far I was not doing it and gels were polymerizing OK but with inconsistent results. 
I would like to know if there is a clear and consistent way of getting HYDROGELS. Maybe it has to do with the reagents, I am using homemade 16% PFA and the rest of the reagents are commercial ones.

Thanks in advance, any suggestion would be greatly appreciated.]]>
        </description>
    </item>
    <item>
        <title>0.5% acrylamide concentration, no polymerization?</title>
        <link>https://forum.claritytechniques.org/discussion/119/0-5-acrylamide-concentration-no-polymerization</link>
        <pubDate>Mon, 11 Aug 2014 13:50:01 +0000</pubDate>
        <category>Hydrogel Embedding</category>
        <dc:creator>Tully</dc:creator>
        <guid isPermaLink="false">119@/discussions</guid>
        <description><![CDATA[Has anyone tried embedding with a 0.5% acrylamide concentration? I just attempted, but its still just pure liquid. Is this normal, or did I mess up the degassing perhaps?]]>
        </description>
    </item>
    <item>
        <title>Has anyone prepared the hydrogel soln. with 4% PFA?</title>
        <link>https://forum.claritytechniques.org/discussion/139/has-anyone-prepared-the-hydrogel-soln-with-4-pfa</link>
        <pubDate>Thu, 11 Dec 2014 02:29:53 +0000</pubDate>
        <category>Hydrogel Embedding</category>
        <dc:creator>hydrazine</dc:creator>
        <guid isPermaLink="false">139@/discussions</guid>
        <description><![CDATA[If so, did it work out?]]>
        </description>
    </item>
    <item>
        <title>Temperatures above 37 deg C?</title>
        <link>https://forum.claritytechniques.org/discussion/132/temperatures-above-37-deg-c</link>
        <pubDate>Sat, 22 Nov 2014 04:56:07 +0000</pubDate>
        <category>Hydrogel Embedding</category>
        <dc:creator>hydrazine</dc:creator>
        <guid isPermaLink="false">132@/discussions</guid>
        <description><![CDATA[Is it okay if the temp gets above 37 degrees during the embedding step?  I can find any mention about slightly higher temps.  ]]>
        </description>
    </item>
    <item>
        <title>Perfusion in rats</title>
        <link>https://forum.claritytechniques.org/discussion/127/perfusion-in-rats</link>
        <pubDate>Tue, 21 Oct 2014 10:25:08 +0000</pubDate>
        <category>Hydrogel Embedding</category>
        <dc:creator>JAGS</dc:creator>
        <guid isPermaLink="false">127@/discussions</guid>
        <description><![CDATA[I have read that the perfusion in rats can be done with 80-120ml aprox. The 10min/ml rule is followed, or change the perfusion time? Is this amount good? ]]>
        </description>
    </item>
    <item>
        <title>Issue with homemade PFA</title>
        <link>https://forum.claritytechniques.org/discussion/115/issue-with-homemade-pfa</link>
        <pubDate>Wed, 09 Jul 2014 02:45:09 +0000</pubDate>
        <category>Hydrogel Embedding</category>
        <dc:creator>Alex_BainsLab</dc:creator>
        <guid isPermaLink="false">115@/discussions</guid>
        <description><![CDATA[I have read several reports where homemade PFA has been cited as the cause of unreliable polymerisation of the hydrogels. I have also had some problems with homemade PFA that seem to be resolved by ensuring the pH of the PFA is &gt;7. I believe that a low pH could inhibit the thermal initiator but I am not sure. Could this be the case? Or could excess protonation inhibit cross-linking directly?]]>
        </description>
    </item>
    <item>
        <title>Saponin</title>
        <link>https://forum.claritytechniques.org/discussion/62/saponin</link>
        <pubDate>Thu, 03 Oct 2013 23:34:36 +0000</pubDate>
        <category>Hydrogel Embedding</category>
        <dc:creator>jdscott</dc:creator>
        <guid isPermaLink="false">62@/discussions</guid>
        <description><![CDATA[Has anyone used saponin for hydrogel embedding of non-perfused tissues? Has it made a difference in the amount of time needed for the hydrogel to diffuse into the tissue? Have you had problems with bubbles in the tissue? I am clearing mouse livers, and I've been able to clear pretty well, but I would like to start using less hydrogel solution if possible by eliminating the perfusion step. Thanks!]]>
        </description>
    </item>
    <item>
        <title>Gel collapsed =(</title>
        <link>https://forum.claritytechniques.org/discussion/95/gel-collapsed</link>
        <pubDate>Mon, 24 Feb 2014 13:51:03 +0000</pubDate>
        <category>Hydrogel Embedding</category>
        <dc:creator>Varf</dc:creator>
        <guid isPermaLink="false">95@/discussions</guid>
        <description><![CDATA[I left freshly embedded samples in vacuum at 37 degrees over 2 days and of course they dried out =( Hydrogel lost around 400% of it's volume and now it's like 2mm thick firm coating the sample. I put it in PBS but it didn't recover. However, it still allows water solution to go through and the gel is not hard or fragile. Now I don't really know if I should proceed with cleaning or it's better to prepare new samples. If it were samples for let's say paraffin IHC I'd throw them out but with hydrogel maybe it's not so crucial? ]]>
        </description>
    </item>
    <item>
        <title>How long can I leave sample before polymerization?</title>
        <link>https://forum.claritytechniques.org/discussion/85/how-long-can-i-leave-sample-before-polymerization</link>
        <pubDate>Wed, 08 Jan 2014 00:51:39 +0000</pubDate>
        <category>Hydrogel Embedding</category>
        <dc:creator>holly</dc:creator>
        <guid isPermaLink="false">85@/discussions</guid>
        <description><![CDATA[Hi,

I was wondering if there is a maximum time that we can leave the sample after we commence perfusion - post fixing - but before we begin polymerization? ]]>
        </description>
    </item>
    <item>
        <title>Loss of GFP Signal</title>
        <link>https://forum.claritytechniques.org/discussion/32/loss-of-gfp-signal</link>
        <pubDate>Tue, 23 Jul 2013 16:09:15 +0000</pubDate>
        <category>Hydrogel Embedding</category>
        <dc:creator>LauraLynch</dc:creator>
        <guid isPermaLink="false">32@/discussions</guid>
        <description><![CDATA[Hi, folks.

When we were first getting started with the CLARITY technique, we perfused a lot of brains at once and left them in the hydrogel at 4 degrees until we were ready to embed them.  When we switched to brains that had been injected with GFP-labeled PRV and to Thy1-EGFP brains, we did the same thing.  What we've been seeing is very good structural preservation, a load of autofluorescence, and no GFP signal at all.  We're assuming that overfixation (beyond the 2 days post-fix in hydrogel as stated in the published protocol) is the cause of our signal loss.  Has anyone else experienced this?  ]]>
        </description>
    </item>
    <item>
        <title>Hydrogel polymerized in the fridge while thawing...</title>
        <link>https://forum.claritytechniques.org/discussion/75/hydrogel-polymerized-in-the-fridge-while-thawing</link>
        <pubDate>Tue, 19 Nov 2013 21:10:14 +0000</pubDate>
        <category>Hydrogel Embedding</category>
        <dc:creator>jroc093</dc:creator>
        <guid isPermaLink="false">75@/discussions</guid>
        <description><![CDATA[Has anyone else experienced this?

I made the hydrogel a month ago and kept everything on ice like the protocol suggested. When it was mixed, I stored the hydrogel in the freezer in 50mL falcon tubes. Now when I go to use them, I put the tubes in the fridge to thaw overnight and there is a gel forming on the bottom of the tube. My fridge is at 4oC.

When I used the hydrogel fresh for the first time (never freezer storage), there was no gel until I put it into my 37oC water bath. Polymerization worked perfectly then.

Thoughts?]]>
        </description>
    </item>
    <item>
        <title>Stopping before ETC step/ Focus Clear</title>
        <link>https://forum.claritytechniques.org/discussion/60/stopping-before-etc-step-focus-clear</link>
        <pubDate>Fri, 27 Sep 2013 09:21:54 +0000</pubDate>
        <category>Hydrogel Embedding</category>
        <dc:creator>Shashank20</dc:creator>
        <guid isPermaLink="false">60@/discussions</guid>
        <description><![CDATA[Hi,

I am at the washing step just before the ETC . Due to some problems with the ETC chamber I need to stop for a week time. Can anyone suggest me  what would be the preferred storage temperature  37oC or 4oC  ?  

Can one suggest a provider of Focus Clear in Denmark/Sweden ? 

Thanks !]]>
        </description>
    </item>
    <item>
        <title>Quick question on the hydrogel solution- PFA fixation concentration</title>
        <link>https://forum.claritytechniques.org/discussion/47/quick-question-on-the-hydrogel-solution-pfa-fixation-concentration</link>
        <pubDate>Thu, 08 Aug 2013 19:11:18 +0000</pubDate>
        <category>Hydrogel Embedding</category>
        <dc:creator>mmokkara</dc:creator>
        <guid isPermaLink="false">47@/discussions</guid>
        <description><![CDATA[We are using CLARITY to visualize lungs/lymph nodes tissue. The lab uses a lower concentration of PFA when fixing the lymph node/lung tissue. I'm wondering if its necessary to use the recommended 16% PFA. I feel that the protocol works best for brain tissues rather than other organs.

Thanks so much for everything!!]]>
        </description>
    </item>
    <item>
        <title>perfusion</title>
        <link>https://forum.claritytechniques.org/discussion/22/perfusion</link>
        <pubDate>Wed, 26 Jun 2013 18:09:24 +0000</pubDate>
        <category>Hydrogel Embedding</category>
        <dc:creator>scottyler89</dc:creator>
        <guid isPermaLink="false">22@/discussions</guid>
        <description><![CDATA[I just tried the clearing on my first sample, which was pancreas from an animal that someone else was already sacrificing, but their sample meant that I couldn't do the PBS and hydrogel perfusion.  Everything seemed to go smoothly regardless though up until the end of my second day clearing, when a vein or artery that had had coagulated blood in it still literally exploded, releasing iron into the media, turning it yellow, likely from iron oxide, which then plated onto the negative electrode.  Just a heads up for anyone else who was straying from the protocol.]]>
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