Journal: International Journal of Molecular Sciences

Article Title: Alteration of Extracellular Matrix Components in the Anterior Pituitary Gland of Neonatal Rats Induced by a Maternal Bisphenol A Diet during Pregnancy

doi: 10.3390/ijms222312667

Figure Lengend Snippet: Immunohistochemistry of NG2 on postnatal Day 1 in the anterior pituitary glands of control rats (( A , C ): higher magnification view of ( A )) and BPA-treated rats (( B , D ): higher magnification view of ( B )). The number ( E ) and mean intensity ( F ) of NG2-positive cells decreased in the BPA-treated group. Note: the capillary lumens (asterisks), * p < 0.05, ** p < 0.01. Scale bars: 100 µm ( A , B ); 10 µm ( C , D ).

Article Snippet: Anti-NG2 rabbit polyclonal antibody (pericyte marker, diluted 1:600; Cat. No. AB5320, Merck Millipore, Darmstadt, Germany), anti-S100 rabbit polyclonal antibody (FS cell marker, diluted 1:1000; Cat. No. Z0311, Agilent DAKO, Santa Clara, CA, USA), anti-aldolase C rabbit polyclonal antibody (novel FS cell marker, diluted 1:200; Cat. No. AB_2571658, Frontier Institute Co., Ltd., Hokkaido, Japan) or anti-collagen type I rabbit polyclonal antibody (diluted 1:400; Cat. No. ab34710, Abcam, Cambridge, UK) were incubated overnight at room temperature.

Techniques: Immunohistochemistry, Control

Journal: Acta neuropathologica

Article Title: Microglia contribute to normal myelinogenesis and to oligodendrocyte progenitor maintenance during adulthood

doi: 10.1007/s00401-017-1747-1

Figure Lengend Snippet: Essential role of microglia for adequate induction of oligodendrocyte progenitors. a Scheme of experimental setup. Cx3cr1GFP/Wt mice were injected intraperitoneally (i.p.) with BLZ945 at P2, P4, P6, and P7. Analysis was performed at P8. b, c Representative blots and quantification of flow cytometric analysis of CD45+CD11b+ microglia (b) and PDGFRα+ oligodendrocyte progenitors (OPCs; c) pre-gated on living cells, single cells, Gr1− cells of mice treated as depicted in a (n = 5–6, samples are from four independent experiments). Each symbol represents one mouse. Means with SEM are represented. Significant differences were determined by an unpaired t test: *P < 0.05, **P < 0.01. d Quantitative RT-PCR of Csf1r of sorted CD45+CD11b+ microglia and NG2+PDGFRα+ OPCs from P8 NG2YFP/Wt mice (n = 5). Data are normalized to Gapdh and β-Actin and presented as fold change to microglia levels. e Representative image of a primary postnatal OPC culture showing overlapping Olig2+/DAPI+ cells (left). OPC cultures were treated for 3 days with vehicle or different concentrations of BLZ945 (0.5, 1, and 5 μM). The number of DAPI+OPCs/coverslip did not change after treatment compared to the vehicle controls. f Representative immunofluorescent images (left) of the corpus callosum showing CX3CR1+ (green) microglia and PDGFRα+ (red) OPCs in vehicle or BLZ945-treated mice. Right quantification of these cells in the corpus callosum (CC), cortex (Cx), and cerebellum (Cb). Each symbol represents one mouse. Means with SEM are represented. n = 4; samples from two independent experiments. Scale bar 40 μm. Significant differences were examined by an unpaired t test and marked with asterisks (*P < 0.05, **P < 0.01). g Representative immunofluorescent images (left) of the corpus callosum showing Sox9+ (red) astrocytes and PDGFRα+ (green) OPCs in vehicle or BLZ945-treated mice. Right quantification of Sox9+/PDGFRα− astrocytes in the corpus callosum (CC), cortex (Cx), and cerebellum (Cb). n = 4–5; samples from two independent experiments. Scale bar 40 μm. Significant differences were examined by an unpaired t test. n.s. not significant. h Quantification of PDGFRα+ OPCs and CC1+ oligodendrocytes in the corpus callosum (CC) and cortex (Cx) of P20 Csf1r+/+ and Csf1r−/− mice. Significant differences were examined by an unpaired t test and marked with asterisks (*P < 0.05). n.s. not significant

Article Snippet: For immunofluorescence in Sox10-iCreER T2 brains, coronal sections (30 μm) were cut and stained as described [ 71 ], with the following primary antibodies: chick-GFP (1:1000, Cat. No. GFP-1020, Aves Lab, Tigard, Oregon, USA), rabbit (rb)-NG2 (1:500, Cat. No. AB5320, Merck Millipore, Darmstadt, Germany), rb-Iba1 (1:500, Cat. No. 01919741, Wako, Neuss, Germany), m-S100β (1:250, Cat. No. S2532, Sigma-Aldrich, Taufkirchen, Germany), and m-GFAP (1:500, Cat. No. G3893, Sigma-Aldrich, Taufkirchen, Germany).

Techniques: Injection, Quantitative RT-PCR

Journal: Acta neuropathologica

Article Title: Microglia contribute to normal myelinogenesis and to oligodendrocyte progenitor maintenance during adulthood

doi: 10.1007/s00401-017-1747-1

Figure Lengend Snippet: Reduced postnatal myelinogenesis in microglia-depleted mice. a Scheme of experimental setup. Cx3cr1GFP/Wt mice were injected intraperitoneally (i.p.) with BLZ945 at P2, P4, P6, and P7. Analysis was done at P20. b–f Quantification of CX3CR1+ microglia (b), PDGFRα+ OPCs (c), CC1+ oligodendrocytes (d), Sox9+/PDGFRα− astrocytes (e), and NeuN+ neurons (f) in the indicated brain regions using immunohistochemically stained brain slices. Significant differences were examined by an unpaired t test and marked with asterisks (*P < 0.05, **P < 0.01). Each symbol represents one mouse. Bars represent mean ± SEM. g Quantitative RT-PCR of the genes proteolipid protein (Plp), myelin basic protein (Mbp), myelin oligodendrocyte glycoprotein (Mog), chondroitin sulfate proteoglycan 4 (Cspg4 = NG2), platelet derived growth factor receptor alpha (Pdgfrα), colony stimulating 1 receptor (Csf1r), and allograft inflammatory factor 1 (Aif1) in tissue isolated from the corpus callosum and cerebellum at P20 (n = 4; samples from two independent experiments). Data were normalized to Gapdh and β-actin and are presented as fold change normed to vehicle. Significant differences were examined by an unpaired t test and marked with asterisks (*P < 0.05, **P < 0.01, ***P < 0.001). h, i Western blot analysis for the myelin proteins proteolipid protein (PLP) and myelin basic protein (MBP) in the corpus callosum and cerebellum at P20. GAPDH was used as a loading control. Quantification (right) was performed by normalization of the myelin proteins to GAPDH. Significant differences were examined by an unpaired t test and marked with asterisks (*P < 0.05, **P < 0.01, ***P < 0.001). Bars indicate mean ± SEM. j Representative electron microscope images from the corpus callosum treated with vehicle or BLZ945 at P20 (left, scale bar 2 μm) and quantification thereof (right). The total number of axons (myelinated and non-myelinated cut off ≥0.5 μm), the percentage of myelinated axons of all axons, and the G-ratio (axon diameter/filament diameter) were quantified (n = 5–6; samples from two independent experiments). Each symbol represents one mouse. Mean ± SEM are presented. Significant differences were examined by an unpaired t test and marked with asterisks (*P < 0.05). k Scheme of experimental setup. Cx3cr1GFP/Wt mice were injected i.p. with BLZ945 at P2, P4, P6, and P7. Analysis was done at P40–42. l Western blot analysis for the myelin proteins PLP and MBP in the corpus callosum and cerebellum at P40–42. GAPDH was used as a loading control. Quantification (right) was performed by normalization of the myelin proteins to GAPDH. Significant differences were examined by an unpaired t test and marked with asterisks (*P < 0.05). Bars indicate mean ± SEM

Article Snippet: For immunofluorescence in Sox10-iCreER T2 brains, coronal sections (30 μm) were cut and stained as described [ 71 ], with the following primary antibodies: chick-GFP (1:1000, Cat. No. GFP-1020, Aves Lab, Tigard, Oregon, USA), rabbit (rb)-NG2 (1:500, Cat. No. AB5320, Merck Millipore, Darmstadt, Germany), rb-Iba1 (1:500, Cat. No. 01919741, Wako, Neuss, Germany), m-S100β (1:250, Cat. No. S2532, Sigma-Aldrich, Taufkirchen, Germany), and m-GFAP (1:500, Cat. No. G3893, Sigma-Aldrich, Taufkirchen, Germany).

Techniques: Injection, Staining, Quantitative RT-PCR, Derivative Assay, Isolation, Western Blot, Microscopy

Journal: Acta neuropathologica

Article Title: Microglia contribute to normal myelinogenesis and to oligodendrocyte progenitor maintenance during adulthood

doi: 10.1007/s00401-017-1747-1

Figure Lengend Snippet: Microglia are necessary for myelin homeostasis during adulthood. a Scheme of experimental setup. 6–10-week-old Cx3cr1GFP/Wt mice were treated with BLZ945 for 7 consecutive days by oral gavage. Analysis was performed 1 day after the last application. b, c Quantification of CX3CR1+ microglia, PDGFRα+ OPCs, CC-1+ oligodendrocytes, Sox9+ astrocytes (b, d) in the corpus callosum (b) and cerebellum (c) using immunohistochemically stained brain sections at day 8. n = 4–7; samples from two independent experiments. Significant differences were examined by an unpaired t test and marked with asterisks (*P < 0.05, **P < 0.01, ***P < 0.001; n.s not significant.) Each symbol represents one mouse. Mean ± SEM are depicted. d Quantitative RT-PCR of the genes proteolipid protein (Plp), myelin basic protein (Mbp), myelin oligodendrocyte glycoprotein (Mog), chondroitin sulfate proteoglycan 4 (Cspg4), platelet derived growth factor receptor alpha (Pdgfrα), colony stimulating 1 receptor (Csf1r), and allograft inflammatory factor 1 (Aif1) in tissue isolated from the corpus callosum and cerebellum at day 8 (n = 4–7; samples from two independent experiments). Data were normalized to Gapdh and β-actin and are presented as fold change normed to vehicle. Significant differences were examined by an unpaired t test and marked with asterisks (*P < 0.05, **P < 0.01, ***P < 0.001). Bars represent mean ± SEM. e Western blot analysis for proteolipid protein (PLP) and myelin basic protein (MBP) in the corpus callosum at P8, 1 day after the last BLZ945 application. GAPDH was used as a loading control. Quantification (right) was performed by normalization of the myelin proteins to GAPDH. Bars indicate mean ± SEM. Significant differences were examined by an unpaired t test (n.s not significant). f Scheme of experimental setup. 8–10-week-old Sox10-iCreERT2 × CAG-eGFP mice were treated with BLZ945 for 5 consecutive days by oral gavage. Analysis was performed 1 day after the last application. g, i Quantification of Iba-1+ microglia, Sox10-GFP+/NG2+ OPCs (g), Sox10-GFP+/NG2− oligodendrocytes and GFAP+/S100β+ astrocytes (i) in the corpus callosum using immunohistochemically stained brain sections at day 6. n = 5–6, samples from two independent experiments. Significant differences were examined by an unpaired t test and marked with asterisks (**P < 0.01, ***P < 0.001; n.s not significant.) Each symbol represents one mouse. Mean ± SEM are depicted. h Representative confocal images of a NG2-immunofluorescence in the corpus callosum of a mouse treated with vehicle (left) or BLZ945 for 5 consecutive days. Scale bar 50 μm

Article Snippet: For immunofluorescence in Sox10-iCreER T2 brains, coronal sections (30 μm) were cut and stained as described [ 71 ], with the following primary antibodies: chick-GFP (1:1000, Cat. No. GFP-1020, Aves Lab, Tigard, Oregon, USA), rabbit (rb)-NG2 (1:500, Cat. No. AB5320, Merck Millipore, Darmstadt, Germany), rb-Iba1 (1:500, Cat. No. 01919741, Wako, Neuss, Germany), m-S100β (1:250, Cat. No. S2532, Sigma-Aldrich, Taufkirchen, Germany), and m-GFAP (1:500, Cat. No. G3893, Sigma-Aldrich, Taufkirchen, Germany).

Techniques: Staining, Quantitative RT-PCR, Derivative Assay, Isolation, Western Blot, Immunofluorescence

Journal: The Journal of Cell Biology

Article Title: NG2-expressing cells in the subventricular zone are type C–like cells and contribute to interneuron generation in the postnatal hippocampus

doi: 10.1083/jcb.200311141

Figure Lengend Snippet: Grafted NG2 + /EGFP + cells differentiate to neurons in the hippocampus. (A–C) 3 wk after transplantation, all the grafted EGFP + cells (green) in the hippocampus are labeled with TUJ1 (red) antibodies. EGFP + /TUJ1 + cells (green/red, respectively) are found in the pyramidal layer of the CA1 (A1) and CA3 (B1). Grafted cells are also found in the hilar region of the DG (C1). (D) A large percentage of transplanted EGFP + cells (green) are also labeled with anti-NeuN (blue) antibodies in the CA3 area and hilar region of the DG (not depicted), confirming the neural fate of the grafted NG2 + /EGFP + cells. The tissue was also stained with anti-GFP antibodies (D3, red), confirming that these neurons are derived from the transplanted NG2 + /EGFP + fraction. (E–G) Neurons derived from grafted NG2 + /EGFP + cells are viable, as determined by TUNEL assay. (E) Positive control (Dnase I-treated tissue). EGFP + /TUJ1 + (F, green/blue, respectively) and EGFP + /NeuN + (G, green/blue, respectively) grafted cells are TUNEL-negative (red) 6 wk after transplantation. Arrows in A1–D1 indicate EGFP + neurons derived from grafted cells. Arrows in E1 indicate EGFP + /TUNEL + cells after DNAse treatment of the tissue (positive control). Arrows in G1 indicate endogenous EGFP-negative TUNEL + cells in DG. EGFP + cells in boxed areas are shown at higher magnification. Bars: (A–D , F, and G) 50 μm; (E) 100 μm.

Article Snippet: An anti-GFP rabbit polyclonal antibody (IgG; 1:250; BD Biosciences) was used to confirm NG2 + cell differentiation by immunohistochemistry.

Techniques: Transplantation Assay, Labeling, Staining, Derivative Assay, TUNEL Assay, Positive Control

Journal: The Journal of Cell Biology

Article Title: NG2-expressing cells in the subventricular zone are type C–like cells and contribute to interneuron generation in the postnatal hippocampus

doi: 10.1083/jcb.200311141

Figure Lengend Snippet: Grafted NG2 + /EYFP + cells give rise to GABAergic interneurons in the hippocampus. (A1–A3) Typical sorting profile for NG2 + /EYFP + cells (NG2 immunolabeling was scattered with RPE-Cy5 [R2 box]). (A4–A6) A typical sample of NG2 + /EYFP + cells after FACS ® shows high purity. (B) 1 wk after grafting, EYFP + cells (green) were found in the hippocampus (arrows) displaying a typical neuronal morphology in the stratum oriens of the CA1 (B3) and CA3 (not depicted), in the stratum pyramidale of the CA1 and CA3 (B2 and B4, respectively), and in the DG (B5). The tissue was also stained with anti-GFP antibodies (C, red), confirming that these neurons are derived from the transplanted NG2 + /EYFP + fraction. Migratory EYFP + cells acquired immature neuronal markers 1 wk after grafting, as shown by Dlx (D, red) and TUJ1 (not depicted). (E and F) 3 wk after transplantation, all the grafted EYFP + cells (green) found in the hippocampus are labeled with anti-NeuN antibodies (blue). EYFP + /NeuN + cells (green/blue) are found in the pyramidal layer of the CA1 (not depicted) and CA3 (E) and in the subgranular layer of the DG (F). EYFP + cells are also labeled with anti–GAD-67 (red) antibodies in the CA3 area (E) and DG (F), confirming their neural fate. In E6 and F6, EYFP was converted to grayscale. Anti-GFP antibodies recognize all the GFP variants, including EGFP, EYFP, and ECFP. EYFP + cells are shown at higher magnification in boxed areas. Bars: (A, C, E, and F) 50 μm; (B1) 300 μm; (B2–B5) 100 μm.

Article Snippet: An anti-GFP rabbit polyclonal antibody (IgG; 1:250; BD Biosciences) was used to confirm NG2 + cell differentiation by immunohistochemistry.

Techniques: Immunolabeling, Staining, Derivative Assay, Transplantation Assay, Labeling