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REAGENTS/MATERIAL:
Serial coronal sections through the rostro-caudal extent of the mouse hippocampus were generated and sections were processed for BrdU immunohistochemistry. In brief, following DNA denaturation and acid hydrolysis, sections were incubated overnight with mouse monoclonal anti-BrdU (Boehringer Mannheim Roche, USA). For immunohistochemical and immunofluorescent detection of endogenous markers of proliferation, immature neurons and the neurogenic transcription factor, tissue sections were exposed to specific primary antibodies:
mouse monoclonal anti-PCNA (Accurate Biochemicals, USA);
goat anti-DCX (Santa-CruzBiotechnology, USA);
mouse monoclonal anti-PSA-NCAM (kind gift from Prof.T.Seki, Juntendo University, Japan);
rabbit anti-TUC-4 (Chemicon, USA) and
goat anti-Neuro D (Santa-Cruz Biotechnology, USA).
We performed studies to detect colocalization of BrdU and GFAP, a marker observed in quiescent hippocampal stem cells, and for BrdU with DCX, a marker observed in proliferating neuroblasts. Sections were incubated with a cocktail of primary antibodies:
mouse monoclonal anti-BrdU (Boehringer Mannheim Roche)
goat anti-DCX (Santa-Cruz Biotechnology, USA) and
rabbit anti-GFAP (Chemicon, USA)
We carried out triple immunofluorescence labeling to detect the neuronal or glial differentiation of BrdU-positive progenitors. Sections were incubated with a cocktail of primary antibodies:
rat anti-BrdU (1:500, Accurate Biochemicals, USA)
mouse monoclonal anti-NeuN (1:1000,Chemicon, USA) and
rabbit anti-GFAP (1:500,Chemicon, USA).
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