Deep antibody penetration in CLARITY-processed tissue, using proteinase K

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, 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?


  • VarfVarf Posts: 13
    edited February 2014
    Going to try Proteinase K and freeze-thaw next week. Will post here observations.
  • VarfVarf Posts: 13
    edited February 2014
  • We'd be very interested in finding out about this modification to the protocol. Can you perhaps send us some more details about where this talk was given?
  • FabioFabio Posts: 62
    Should be this lecture
    17th Kenneth B Myer Lecture - Dr. Karl Deisseroth
  • the only thing i know about the talk is what is shown in the youtube-video, posted by Fabio.
    sounds very interesting Varf! looking forward to hear about your progress!
  • jdscottjdscott Posts: 8
    I am also considering using these methods and would be interested in hearing your results! Are you doing the treatments before or after clearing, and have you tried with any other clearing method such as SeeDB?
  • KeithSiewKeithSiew Posts: 24
    Thanks Fabio. The question and answer is at 1hr 6mins 34s

    Also you got yourself a name drop by Deisseroth "The Fabio"


    We're going to try the Proteinase step and see where it gets us
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