Penetration depth of antibody


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?


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    Hi Iijun,
    I am also trying to image post mortem tissue with antibodies, and I didn't get my lucky shot yet. As stated in the article, a two days incubation of first antibody should suffice for imaging. The supplementary video included in the article shows a good overall staining in human brain. However, with an increase of incubation or with an increase of antibody concentration, I did not manage to reproduce these results.
    Are there people here with working antibody stainings on post mortem human tissue? I do get the feeling that antibodies might be too large to pass the hydrogel barrier, because my RedDot stainings do work.
    Hope somebody has suggestions here, cheers!
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    We're about to move on to antibody staining of our human and mouse tissues so this is a concern for us also.

    The authors said that 1mm for 3days per antibody and 14days for 5mm was what was needed for homogenous complete staining. Have you tried these extended times? We've been thinking about including higher concentrations of Triton X-100 and/or Saponin to see if it will assist us.

    The time for diffusion should be T = (L^2)/(2*D), where T is time, L is the distance to diffuse across and D is the diffusion coefficient of the molecule in the system. A cancer immunotherapy paper by Jain & Baxter showed that in tumours (which are notoriously difficulty to penetrate) the time it takes a typical IgG macromolecule to diffuse through 1mm of tissue is ~54hrs and it should be noted that the relationship of diffusion time is proportional the to square of distance so the time becomes exponentially longer for deeper staining. Granted these are in vivo scenarios and exploiting vasculature to deliver the antibodies but this should give us some clues.

    Another thread has suggested the use of Methnol/Freeze-thaw cycles/Proteinase K steps to increase penetration, which is currently being used in the SeeDB technique
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    irinairina Posts: 6
    i was wondering what volume of antibody solution are people using for their stainings since the antibody dilution is just 1:50 to 1:100
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    We started with 1:200, and seems to work fine (though I am sure we can thin it a bit more). We also did a 24hr and 48hr incubations (@37oC) and did not notice a difference in 2mm, 1mm and 500um thick rat brain. Really the only modification (in SeeDB) that seemed to improve IHC was the freeze-thaw (We tried several, combinations, and F/T was the one that seemed to do it for us). But remember that all of these manipulations are done PRIOR to SeeDB, whereas in CLARITY it seems that (at least the IHC) they would have to occur after the CLARITY procedure.
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    irinairina Posts: 6
    I managed to get clarity work on 1mm thick mouse slice, 1ml antibody solution 1:100 (however high background). I tried to imagine a rat slice but the signal is very weak so I was wondering how much more antibody solution should be used
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    helenhelen Posts: 4
    I am using ~2-3mm mouse brain slices, 0.5ml antibody solutions of 1:100-1:200 dilution, and 1 week incubation times. I'm still working on my imaging quality through the z-stack, but can see antibody penetration to ~1.5mm fairly routinely.
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    lorenzoolorenzoo Posts: 2
    @helen‌ and @irina‌, which antibodies are you using?
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    VarfVarf Posts: 13
    Hi everyone, I tried Proteinase K 20µg/ml and 1mg/ml, incubation at 37 degrees for 10 min. Pryor to it I did Methanol freeze/thaw cycles. After 1mg I had even no dapi staining and tissue looked damaged. After 20µg I didn't see any improvements compare to Methanol only. However, Methanol only was better in terms of a/b penetration then without Methanol at all. I don't have a confocal, but in my case Methanol increased the depth of visible stained area from 10 to 25-30µm.
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