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        <title>Sample Staining — CLARITY forum</title>
        <link>https://forum.claritytechniques.org/</link>
        <pubDate>Sat, 08 Aug 2026 01:16:45 +0000</pubDate>
        <language>en</language>
            <description>Sample Staining — CLARITY forum</description>
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    <item>
        <title>How to store the immunostained sample after imaging?</title>
        <link>https://forum.claritytechniques.org/discussion/200/how-to-store-the-immunostained-sample-after-imaging</link>
        <pubDate>Wed, 21 Jun 2017 20:07:44 +0000</pubDate>
        <category>Sample Staining</category>
        <dc:creator>xyang</dc:creator>
        <guid isPermaLink="false">200@/discussions</guid>
        <description><![CDATA[Hello,

I am starting to try CLARITY and immunostaining on my 1-mm-thick mouse brain slices. I am wondering how do people store their immunostained samples after imaging in case they are needed for a second time imaging sometime later? I am concerned that if storing in PBS or clearing solution will let antibodies diffuse out over time and if that means the sample can only be imaged within a short period of time after immnostaining? Are there any tricks to prevent antibodies from leaking out of the sample?

Thanks!]]>
        </description>
    </item>
    <item>
        <title>Antibodies used in CLARITY literature</title>
        <link>https://forum.claritytechniques.org/discussion/203/antibodies-used-in-clarity-literature</link>
        <pubDate>Thu, 11 Jan 2018 19:07:03 +0000</pubDate>
        <category>Sample Staining</category>
        <dc:creator>Kristin_Engberg</dc:creator>
        <guid isPermaLink="false">203@/discussions</guid>
        <description><![CDATA[Hi everyone,

In case you're looking for antibodies that will work well in your CLARITY samples, the CLARITY wiki now has a table of primary antibodies that have been used/tested on CLARITY samples in the published literature: http://wiki.claritytechniques.org/index.php/Immunostaining]]>
        </description>
    </item>
    <item>
        <title>Anti-GFAP in CLARITY</title>
        <link>https://forum.claritytechniques.org/discussion/155/anti-gfap-in-clarity</link>
        <pubDate>Wed, 25 Mar 2015 12:51:18 +0000</pubDate>
        <category>Sample Staining</category>
        <dc:creator>jose_jpr</dc:creator>
        <guid isPermaLink="false">155@/discussions</guid>
        <description><![CDATA[I'll do a pilot experiment with a sample of rat. Had thought to use Anti-GFAP, but I don't know the concentration to use it. Has anyone used this antibody?]]>
        </description>
    </item>
    <item>
        <title>Staining Neurons and counting them using CLARITY?</title>
        <link>https://forum.claritytechniques.org/discussion/197/staining-neurons-and-counting-them-using-clarity</link>
        <pubDate>Thu, 06 Apr 2017 20:02:26 +0000</pubDate>
        <category>Sample Staining</category>
        <dc:creator>maryam_z</dc:creator>
        <guid isPermaLink="false">197@/discussions</guid>
        <description><![CDATA[we are using the CLARITY technique to clear Rat brains and we would like to label astrocytes and neurons in the cerebral cortex and to be able to count them. We tried to stain it using NeuN and GFAP, but it appears that it didn’t stain when we did imaging. Do you have any recommendations/thoughts on why it did not work, or any specific steps we should follow? 
we followed steps on 
http://wiki.claritytechniques.org/index.php/Immunostaining]]>
        </description>
    </item>
    <item>
        <title>Dextran and fluoro gold</title>
        <link>https://forum.claritytechniques.org/discussion/191/dextran-and-fluoro-gold</link>
        <pubDate>Wed, 28 Sep 2016 02:18:35 +0000</pubDate>
        <category>Sample Staining</category>
        <dc:creator>cytokine</dc:creator>
        <guid isPermaLink="false">191@/discussions</guid>
        <description><![CDATA[Has anyone used dextran Alexa or fluoro gold  with  clarity? Any issues? Thank you ]]>
        </description>
    </item>
    <item>
        <title>DAPI staining</title>
        <link>https://forum.claritytechniques.org/discussion/133/dapi-staining</link>
        <pubDate>Tue, 25 Nov 2014 13:59:15 +0000</pubDate>
        <category>Sample Staining</category>
        <dc:creator>Tully</dc:creator>
        <guid isPermaLink="false">133@/discussions</guid>
        <description><![CDATA[Hi all,

I have a 2mm thick slice of rat brain, and I'm DAPI-staining it at a 10nM concentration at the moment (because thats the number I kept stumbling across). I've left it for roughly a week now, but I'm unsure how long I should leave it. It appears to vary a lot with staining type.

How long should I leave it, and how long should I wash it? Has anyone tried DAPI?

When I stained for perineuronal nets, I got a lot of unspecific binding on the very surface (not too much background other than that though). Would that be due to insufficient washing perhaps?

Cheers. ]]>
        </description>
    </item>
    <item>
        <title>Fluorescent tracing</title>
        <link>https://forum.claritytechniques.org/discussion/129/fluorescent-tracing</link>
        <pubDate>Wed, 12 Nov 2014 10:49:41 +0000</pubDate>
        <category>Sample Staining</category>
        <dc:creator>JAGS</dc:creator>
        <guid isPermaLink="false">129@/discussions</guid>
        <description><![CDATA[Hi all, I write you, because I want to study the neuronal pathway with tracers using CLARITY.
Which is the best tracer to use in CLARITY?]]>
        </description>
    </item>
    <item>
        <title>Anyone tried second round of staining?</title>
        <link>https://forum.claritytechniques.org/discussion/166/anyone-tried-second-round-of-staining</link>
        <pubDate>Thu, 21 May 2015 17:52:37 +0000</pubDate>
        <category>Sample Staining</category>
        <dc:creator>mhurry</dc:creator>
        <guid isPermaLink="false">166@/discussions</guid>
        <description><![CDATA[The CLARITY wiki states that you can wash out the first antibodies by washing in clearing solution overnight at 60°C. Anyone had any luck with removing the first lot of antibodies and reprobing the tissue?]]>
        </description>
    </item>
    <item>
        <title>PACT and Immunos</title>
        <link>https://forum.claritytechniques.org/discussion/159/pact-and-immunos</link>
        <pubDate>Thu, 02 Apr 2015 00:52:23 +0000</pubDate>
        <category>Sample Staining</category>
        <dc:creator>bpham</dc:creator>
        <guid isPermaLink="false">159@/discussions</guid>
        <description><![CDATA[I have been clearing tissue using the PACT protocol on 1mm sections of mouse spinal cord. I use 4% acrylamide and 8% SDS. 

I incubate in primary for 3 days at room temperature at a concentration of 1:200
Wash in PBS for a day
Incubate in secondary for 3 days at room temperature at 1:200 
Wash in PBS for a day
Put into RIMS for 2 days. 

The tissue is not as transparent as tissue I put into RIMS after the SDS wash and 1 day PBS wash without running immunos.

Has this happened to anyone else?]]>
        </description>
    </item>
    <item>
        <title>immuno CLARITY and sodium azide</title>
        <link>https://forum.claritytechniques.org/discussion/156/immuno-clarity-and-sodium-azide</link>
        <pubDate>Thu, 26 Mar 2015 12:53:22 +0000</pubDate>
        <category>Sample Staining</category>
        <dc:creator>jose_jpr</dc:creator>
        <guid isPermaLink="false">156@/discussions</guid>
        <description><![CDATA[On the protocol CLARITY immuno that the sample can be stored in PBST (with 0.01% (w / v) sodium azide) at 4 ° C for up to a week, but azide and fluorescent antibodies are incompatible or'm wrong. Thanks]]>
        </description>
    </item>
    <item>
        <title>Validated antibodies</title>
        <link>https://forum.claritytechniques.org/discussion/140/validated-antibodies</link>
        <pubDate>Sat, 13 Dec 2014 00:38:48 +0000</pubDate>
        <category>Sample Staining</category>
        <dc:creator>JLM</dc:creator>
        <guid isPermaLink="false">140@/discussions</guid>
        <description><![CDATA[Following some successful passive tissue clearing, I'm ready to start exploring useful antibodies for tissue staining.
As with any fixation method, some antibodies work and some don't.

Can those with successful staining of passively cleared tissues please list here some of their vetted antibodies for CLARITY?
Are there any good vasculature markers out there?  Listing of validated antibodies for ANY target will be helpful, if only for positive controls.
]]>
        </description>
    </item>
    <item>
        <title>Staining ETC samples</title>
        <link>https://forum.claritytechniques.org/discussion/59/staining-etc-samples</link>
        <pubDate>Thu, 26 Sep 2013 09:09:10 +0000</pubDate>
        <category>Sample Staining</category>
        <dc:creator>ClarityNL</dc:creator>
        <guid isPermaLink="false">59@/discussions</guid>
        <description><![CDATA[Has anybody tried to stain samples for imaging yet? I want to try and stain my cleared mouse brain, but don't want to spill my AB's optimizing this. Do any of you have tips regarding staining of thick slides of mouse brain (5 mm slice)?
Cheers, Esther]]>
        </description>
    </item>
    <item>
        <title>Antibody staining for dendritic/spine retraction</title>
        <link>https://forum.claritytechniques.org/discussion/98/antibody-staining-for-dendritic-spine-retraction</link>
        <pubDate>Mon, 03 Mar 2014 20:57:09 +0000</pubDate>
        <category>Sample Staining</category>
        <dc:creator>kristie</dc:creator>
        <guid isPermaLink="false">98@/discussions</guid>
        <description><![CDATA[Hi all, I was just wondering if anyone had any suggestions for me for a protein to stain for looking at dendritic spine retraction in a stress protocol (or any other protocol that induces changes). I have some structural protein ideas in mind, but I am just checking to see if anyone could suggest a protein present/expressed when alterations dendritic morphology occurs (I'm a little new to this field).

Cheers.]]>
        </description>
    </item>
    <item>
        <title>Penetration  depth of antibody</title>
        <link>https://forum.claritytechniques.org/discussion/71/penetration-depth-of-antibody</link>
        <pubDate>Tue, 29 Oct 2013 13:42:56 +0000</pubDate>
        <category>Sample Staining</category>
        <dc:creator>lijun</dc:creator>
        <guid isPermaLink="false">71@/discussions</guid>
        <description><![CDATA[Hi,folks.

The thickness of my brain section is about 500um(the whole brain has been clearing for 1 week with 37℃,25V).And I did the immunostaining with the protocol provided by the authors(primary antibody:1d/washing:12h/  secondary antibody:1d/washing:12h). And follow the staining tips,eg 37℃ on a shaker.

But it was really depressed that the penetration depth of antibody was less than 100um.Even I increased the incubating time for both primary antibody and secondary antibody(3days) ,the depth was only about 100um.

Has anyone else experienced this? or any suggestion?
]]>
        </description>
    </item>
    <item>
        <title>Electro-immunofluorescence staining for better Ab staining of thick tissues--thoughts?</title>
        <link>https://forum.claritytechniques.org/discussion/100/electro-immunofluorescence-staining-for-better-ab-staining-of-thick-tissues-thoughts</link>
        <pubDate>Wed, 12 Mar 2014 16:30:16 +0000</pubDate>
        <category>Sample Staining</category>
        <dc:creator>hutchpd</dc:creator>
        <guid isPermaLink="false">100@/discussions</guid>
        <description><![CDATA[I'm about to start troubleshooting our CLARITY process and came across this paper for achieving very nice antibody staining in whole mount tissues. Granted, their mouse corneas are only 120µm thick (which is nothing compared to whole organs), but the principle they used sounds promising...

Here's the paper:
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2650717/?report=classic

I'm curious, for those who have experience, if you think the cleared tissue samples would tolerate this protocol? Or if an additional embedding/electrophoresis step would just obliterate the sample? Passive diffusion just seems like a losing battle between enough permeabilization for staining versus sample destruction.

Thoughts?]]>
        </description>
    </item>
    <item>
        <title>Deep antibody penetration in CLARITY-processed tissue, using proteinase K</title>
        <link>https://forum.claritytechniques.org/discussion/94/deep-antibody-penetration-in-clarity-processed-tissue-using-proteinase-k</link>
        <pubDate>Wed, 19 Feb 2014 11:03:32 +0000</pubDate>
        <category>Sample Staining</category>
        <dc:creator>pensare</dc:creator>
        <guid isPermaLink="false">94@/discussions</guid>
        <description><![CDATA[chung and deisseroth cite this article in their review "Light microscopy mapping of connections in the intact brain" (SVSNEURO also mentioned it in another discuccion):
A Method for 3D Immunostaining and Optical Imaging of the Mouse Brain Demonstrated in Neural Progenitor Cells" Plos One 8, Published: August 06, 2013 DOI:10.1371/journal.pone.0072039 Jacqueline A. Gleave, Jason P. Lerch, R. Mark Henkelman, Brian J. Nieman

in this article they treat the tissue with a freeze-thaw cycle, ending with a short incubation in proteinase K. they report antibody staining of 1 mm thick tissue samples.

at 1 hour 8 minutes into this lecture deisseroth talks about achieving deeper antibody oenetration, https://www.youtube.com/watch?v=uf-msQ-QJ6A. he mentions that they have started using both proteinase K, and a lower concentration of bis in the monomer-solution, to reduce the cross-linking extent.

has anyone of you guys experimented with proteinase K, lower bis-concentration, or any other protocols in order to achieve deeper antibody penetration?
]]>
        </description>
    </item>
    <item>
        <title>1 M sodium borate buffer solution</title>
        <link>https://forum.claritytechniques.org/discussion/52/1-m-sodium-borate-buffer-solution</link>
        <pubDate>Tue, 20 Aug 2013 04:47:03 +0000</pubDate>
        <category>Sample Staining</category>
        <dc:creator>Txema</dc:creator>
        <guid isPermaLink="false">52@/discussions</guid>
        <description><![CDATA[We have obtained our first partially cleared samples and now we wanted to try immunofluorescence on those samples. We have difficulties dissolving and preparing the 1 M sodium borate buffer solution, pH 8.5. Could somebody post a recipe for preparing it? Thank you in advance.]]>
        </description>
    </item>
    <item>
        <title>Saturation of tissue before staining and buffer of incubation</title>
        <link>https://forum.claritytechniques.org/discussion/73/saturation-of-tissue-before-staining-and-buffer-of-incubation</link>
        <pubDate>Thu, 14 Nov 2013 09:51:45 +0000</pubDate>
        <category>Sample Staining</category>
        <dc:creator>Christelle</dc:creator>
        <guid isPermaLink="false">73@/discussions</guid>
        <description><![CDATA[I want to stain my cleared mouse brain, but I look the protocol described in the publication, there is no saturation of the aspecific site with serum from the 2nd antibody specie. Has anybody tried to stain samples  including this step before the incubation with the first antibody? has anybody tried to incubate the antibodies in other solution that the sodium borate buffer?
Cheers
Christelle]]>
        </description>
    </item>
    <item>
        <title>Immunostaining of CLARITY-processed post-mortem human brain tissue</title>
        <link>https://forum.claritytechniques.org/discussion/82/immunostaining-of-clarity-processed-post-mortem-human-brain-tissue</link>
        <pubDate>Mon, 16 Dec 2013 10:59:45 +0000</pubDate>
        <category>Sample Staining</category>
        <dc:creator>Irene</dc:creator>
        <guid isPermaLink="false">82@/discussions</guid>
        <description><![CDATA[Hello all, 
I would like to reproduce the immunostaining protocol on post-mortem human brain tissue, however on the article I can't find any specification about the antibodies used. Could you please give me any suggestion about where I can buy the first and the second antibody for the tyrosine hydroxylase and parvalbumin staining? 
Thank you!
Irene]]>
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