For Western blot analysis, adult female wild-type and
Upf3b−/− brains were isolated,
chopped into small pieces, and placed into a RIPA lysis buffer (Sigma)
containing phenylmethane sulfonyl fluoride for 30 minutes on ice to lyse cells
and release proteins. Protein concentration was calculated using a DC BSA assay
(Bio-Rad Laboratories) and Western blotting was performed as described
previously28 (link). For
immunohistochemistry, adult male wild-type and Upf3b-null
brains were isolated and immediately placed into optimal-cutting temperature
(OCT [Sakura]) solution and frozen for cryosectioning.
Cryosectioning was done at 12 μm thickness using a Leica 1500 machine
and tissue sections were placed on Superfrost microscope slides (Fisherbrand)
and stored at −80C until ready for use. At the time of staining, the
sections were air-dried for 20 min, followed by incubation with 4%
paraformaldehyde (Electron Microscopy Sciences) for 10 min to fix the tissue.
After PBS washes, the sections were permeabilized with 0.1% Triton-X 100
for 10 min. The subsequent staining procedures were done as previously
described29 (link). mNSCs
immunohistochemistry was performed as described previously31 (link). Microscopy analysis was performed using
a Leica AF6000 epi-fluorescence microscope.
Upf3b−/− brains were isolated,
chopped into small pieces, and placed into a RIPA lysis buffer (Sigma)
containing phenylmethane sulfonyl fluoride for 30 minutes on ice to lyse cells
and release proteins. Protein concentration was calculated using a DC BSA assay
(Bio-Rad Laboratories) and Western blotting was performed as described
previously28 (link). For
immunohistochemistry, adult male wild-type and Upf3b-null
brains were isolated and immediately placed into optimal-cutting temperature
(OCT [Sakura]) solution and frozen for cryosectioning.
Cryosectioning was done at 12 μm thickness using a Leica 1500 machine
and tissue sections were placed on Superfrost microscope slides (Fisherbrand)
and stored at −80C until ready for use. At the time of staining, the
sections were air-dried for 20 min, followed by incubation with 4%
paraformaldehyde (Electron Microscopy Sciences) for 10 min to fix the tissue.
After PBS washes, the sections were permeabilized with 0.1% Triton-X 100
for 10 min. The subsequent staining procedures were done as previously
described29 (link). mNSCs
immunohistochemistry was performed as described previously31 (link). Microscopy analysis was performed using
a Leica AF6000 epi-fluorescence microscope.