Deep antibody penetration in CLARITY-processed tissue, using proteinase K
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Posts: 3
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?
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?
Comments
17th Kenneth B Myer Lecture - Dr. Karl Deisseroth
sounds very interesting Varf! looking forward to hear about your progress!
Also you got yourself a name drop by Deisseroth "The Fabio"
Haha
We're going to try the Proteinase step and see where it gets us