For BrdU immunofluorescence staining, sections were incubated in TBS containing
Triton X-100, blocked with TBS containing
Triton X-100/5% donkey serum then incubated with
monoclonal rat anti-BrdU (Accurate Chemical & Scientific Corporation) and
monoclonal mouse anti-NeuN (Millipore Bioscience Research Reagents) in TBS for overnight at 4°C.
Immunofluorescence and immunoblotting of NPC culture: For immunodetection
of Nestin expression, neurospheres were transferred
onto a slide precoated with poly-L-ornithine (Nalge Nunc) containing serum-free medium.
Four days later, the neurospheres adhering to the slide chamber
were fixed with cold methanol, blocked for 30 min PBS containing Triton X-100 (PBST) and
10% normal horse serum. Samples were washed with PBST and incubated
with mouse monoclonal anti-Nestin (clone R-401) (Dr. D.
Geschwind, University of California at Los Angeles, Los Angeles, CA). For immunoblotting
analysis, neurosphere protein samples were prepared in lysis buffer, fractionated by electrophoresis in
Tris-Tricine polyacrylamide gels, transferred to nitrocellulose
membranes and immunoblotted with antibodies raised against the N-terminus
or loop domains of PS1 (Kim and Sisodia, 2005).
NPC proliferation assays: Proliferation rate of NPCs was measured by
BrdU incorporation using a nonisotopic quantitative immunoassay kit
(Calbiochem). The extent of BrdU-uptake was determined by
fixing the cells, denaturing theDNAand immunostaining for BrdU, with
reagents supplied by the manufacturer. For immunocolabeling with nestin and BrdU, single cell suspension
of primary neurospheres were plated onto a precoated slide in
serum-free medium supplemented with BrdU and cultures were fixed, treated
with HCl followed by borate buffer, blocked with PBST and donkey serum and processed for immunostaining
with anti-nestin and BrdU antibodies as described earlier (Song
et al., 2002;
Choi et al., 2008).
Multipotency of NPCs: Primary neurospheres were dissociated into single
cell suspension individual cells were plated onto a slide containing differentiation inducing medium. Adherent cells were
fixed and processed for immunofluoresence staining to detect lineage specific
antigens: neuronal marker, βIII-tubulin (TUJ1) (Covance Research Products);
astrocyte markers, GFAP (Dako)
and s100β (Swant) and
oligodendrocyte marker, O4 (StemCell Technologies).
Expression of human PS1 polypeptides and their ability to process APP-CTF
substrate were confirmed by transducing PS-deficient fibroblasts
and later cells were lysed and total protein was
subjected for immunoblotting analysis with PS1 NT,
APP 369 or
GFP (Axxora) antibodies.
Expression of Notch1-ICD polypeptide was confirmed by transducing HEK 293 cells and
immunoblotting the lysate with mouse monoclonal anti-HA (12CA) (Roche Bioscience).