Journal: Scientific Reports
Article Title: Olig2 regulates Purkinje cell generation in the early developing mouse cerebellum
Figure Lengend Snippet: Olig gene-expressing progenitors give rise to PCs and DCN neurons but rarely Pax2 + interneurons in the postnatal cerebellum as revealed by long-term lineage tracing. Olig1-Cre lineage tracing analysis is performed in the cerebellum of either Z/EG ( A–H ) reporter line at P18 or ROSA26;LacZ ( I ) reporter line at P16. Labeled cells with different morphology in the cerebellum of Olig1-Cre;Z/EG mice are revealed by their GFP expression ( A ). The identity of GFP + cells are examined by co-staining with cell-type specific markers Calbindin ( B ), Parvalbumin ( C ), GFAP ( D ), BLBP ( E ), Olig2 ( F,G ) and Pax2 ( H ). Some GFP + cells are indicated as either negative (arrows in C and H ) or positive (arrows in F,G ) for marker staining. The high magnification insert in ( C ) confirms the cellular identity of the indicated GFP + cell by showing the presence of DAPI + nucleus. Many labeled cells in the cerebellar DCN of Olig1-Cre;LacZ reporter mice are observed by anti-β-gal staining, some of which are NeuN + (arrow, I ) and GABA + (arrow, insert in I ). Olig2-Cre lineage tracing analysis is also performed in the cerebellum of ROSA26;tdTomato ( J–P ) reporter line at P22. Labeled cells with different morphology in the cerebellum of Olig2-Cre;tdTomato mice are revealed by their tdTomato expression ( J ). The identity of tdTomato + cells are examined by co-staining with cell-type specific markers Calbindin ( K ), Parvalbumin ( L ), GFAP ( M ), Olig2 ( N ), CC1 ( O ) and Pax2 ( P ). Note that two tdTomato + cells are co-labeled with Parvalbumin suggestive of small interneurons (arrows in L ). DAPI is used to label nuclei. ML, molecular layer; IGL, internal granule layer; WM, white matter; DCN, deep cerebellar nuclei. Scale bar: 100 μm in ( A,J ); 20 μm in ( B–H , K–P ); 25 μm in I .
Article Snippet: Tissue sections or fixed cell cultures were incubated with monoclonal antibodies against BrdU (rat IgG, 1:200, Accurate), Olig2 (mouse IgG, 1:200, Millipore), APC (mouse IgG, clone CC-1, 1:200, Calbiochem) and NeuN (mouse IgG, 1:400, Millipore); polyclonal antibodies against β-Galactosidase (β-gal) (chicken IgY, 1:2000, Abcam), BLBP (rabbit IgG, 1:500, Abcam), Tbr1 (rabbit IgG, 1:400, Proteintech), Pax2 (rabbit IgG, 1:50, Proteintech), Pax6 (rabbit IgG, 1:100, Proteintech), GABA (rabbit IgG, 1:2000, Sigma), Parvalbumin (rabbit IgG, 1:800, ImmunoStar), Olig2 (rabbit IgG, 1:500, Millipore or guinea pig IgG, 1:500, a gift from Dr. Ben Novitch at UCLA), GFAP (chicken IgY, 1:1000, Aves or rabbit IgG, 1:2000, DAKO), Calbindin (rabbit IgG, 1:500, ImmunoStar), Calretinin (rabbit IgG, 1:500, ImmunoStar), cleaved caspase-3 (rabbit IgG, 1:1000, Cell Signaling), and DCX (guinea pig IgG, 1:1000, Millipore), followed by appropriate species-specific secondary antibodies (Molecular Probes).
Techniques: Expressing, Labeling, Mouse Assay, Staining, Marker