rb ng2 antibody  (Millipore)

Journal: eLife

Article Title: Activity-dependent modulation of synapse-regulating genes in astrocytes

doi: 10.7554/eLife.70514

Figure Lengend Snippet: ( A ) Analysis of cell-specific genes in HA-pulldown/input samples (log2) from RNA sequencing demonstrates enrichment for astrocyte genes and depletion of other cells in all samples (N = 3 at postnatal day [P]7, 4 at P14, 5 at P28, 3 at P120; for statistical comparisons, 3xP120 samples published in were added to increase the power of the analysis). ( B–D ) Immunostaining for the HA tag and cell-specific markers to determine cell-type expression of tagged ribosomes in P28 visual cortex. ( D ) Representative images, left panels: cell marker; middle panels: HA; right panels: merge with DAPI to mark nuclei. ( B, C ) Quantification of ( D ). ( B ) Quantification of colocalization of HA with each cell-specific marker, expressed as % of marker + cells, demonstrates that majority of HA+ cells are astrocytes. ( C ) Quantification of number of astrocytes (s100β+) that are also HA+ demonstrates that majority of astrocytes express HA-tagged ribosomes. N = 4 mice S100β, NEUN, IBA1; 3 mice MOG; 2 mice NG2. Bar graphs mean ± s.e.m. Scale bars = 20 µm. ( E ) Heatmaps of top 20 most astrocyte-enriched genes (highest astro/IN ratio; FPKM > 100) at each time point, as well as those in the top 20 at all time points, represented as fold change (FC) of astrocyte/input (log2). ( F ) Venn diagram showing overlap in astrocyte-enriched genes at each age. ( G ) Gene Ontology (GO) terms analysis with String db of Biological Process (BP) in astrocyte-enriched genes at each time point. Venn diagram showing overlap in GO terms at each age.

Article Snippet: The following antibodies were used: Chk anti-GFP (Millipore #06-896, 1:500), Rb anti-SOX9 (Abcam #ab185966, 1:2000), Rb anti-ALDH1L1 (Abcam #ab-87117, 1:500), Rb anti-HA (CST #3724), Rb anti-S100β (Abcam #ab52642, 1:100), Ms anti-NEUN (Millipore #MAB377 1:100), Rb anti-NG2 (Millipore # Ab5320), Rb anti-MOG (Proteintech # 12690-1-ap), Rb anti-IBA1 (Wako #016-20001), Gp anti-VGLUT1 (Millipore #AB5905, 1:2000), Gp anti-VGLUT2 (Millipore #AB2251 1:3000, 1:5000), Rb anti-GLUA1 (Millipore #AB1504, 1:400), Rb anti-GLUA2 (Millipore #AB1768-I, 1:400), and Ms anti-Bassoon (Enzo #VAMP500, 1:500).

Techniques: RNA Sequencing Assay, Immunostaining, Expressing, Marker

Journal: eLife

Article Title: Activity-dependent modulation of synapse-regulating genes in astrocytes

doi: 10.7554/eLife.70514

Figure Lengend Snippet:

Article Snippet: The following antibodies were used: Chk anti-GFP (Millipore #06-896, 1:500), Rb anti-SOX9 (Abcam #ab185966, 1:2000), Rb anti-ALDH1L1 (Abcam #ab-87117, 1:500), Rb anti-HA (CST #3724), Rb anti-S100β (Abcam #ab52642, 1:100), Ms anti-NEUN (Millipore #MAB377 1:100), Rb anti-NG2 (Millipore # Ab5320), Rb anti-MOG (Proteintech # 12690-1-ap), Rb anti-IBA1 (Wako #016-20001), Gp anti-VGLUT1 (Millipore #AB5905, 1:2000), Gp anti-VGLUT2 (Millipore #AB2251 1:3000, 1:5000), Rb anti-GLUA1 (Millipore #AB1504, 1:400), Rb anti-GLUA2 (Millipore #AB1768-I, 1:400), and Ms anti-Bassoon (Enzo #VAMP500, 1:500).

Techniques: Plasmid Preparation, Recombinant, Protease Inhibitor, Bradford Assay, Sequencing, Negative Control, Software, Cell Culture, Fluorescence, Microscopy, Hybridization

Journal: eLife

Article Title: Activity-dependent modulation of synapse-regulating genes in astrocytes

doi: 10.7554/eLife.70514

Figure Lengend Snippet:

Article Snippet: The following antibodies were used: Chk anti-GFP (Millipore #06-896, 1:500), Rb anti-SOX9 (Abcam #ab185966, 1:2000), Rb anti-ALDH1L1 (Abcam #ab-87117, 1:500), Rb anti-HA (CST #3724), Rb anti-S100β (Abcam #ab52642, 1:100), Ms anti-NEUN (Millipore #MAB377 1:100), Rb anti-NG2 (Millipore # Ab5320), Rb anti-MOG (Proteintech # 12690-1-ap), Rb anti-IBA1 (Wako #016-20001), Gp anti-VGLUT1 (Millipore #AB5905, 1:2000), Gp anti-VGLUT2 (Millipore #AB2251 1:3000, 1:5000), Rb anti-GLUA1 (Millipore #AB1504, 1:400), Rb anti-GLUA2 (Millipore #AB1768-I, 1:400), and Ms anti-Bassoon (Enzo #VAMP500, 1:500).

Techniques: Marker

Journal: Frontiers in Cell and Developmental Biology

Article Title: NG2-Glia Transiently Overcome Their Homeostatic Network and Contribute to Wound Closure After Brain Injury

doi: 10.3389/fcell.2021.662056

Figure Lengend Snippet: Temporal reaction of NG2-glia after injury. (a–c) Images of NG2-glia around the injury site at d0 (a) , d4 (b) , and d28 (c) after PWI. (d,e,g,h) Graphs depict the percentage (mean + SEM) of cells showing hypertrophy, polarization, proliferation and migration at the given timepoints (n = 3 − 8 animals per timepoint). “New” (green bars) represent the cells showing hypertrophy (d) , polarization (e) , proliferation (g) and migration (h) for the first time at the indicated timepoint. “Old” (red bars) represent cells which showed this behavior already at the previous timepoint. (f,i) Directionality of polarized (f) or migrating (i) cells (mean + SEM; yellow bars represent the percentage of polarized cells with a direction towards the quadrant enclosing the PWI; grey bars represent percentage of polarized cells towards the remaining 3 quadrants) over time. n = 8 mice for timepoint d2, n = 6 mice for timepoint d4, n = 4 mice for timepoint d8, n = 3 mice for all other timepoints; mostly 20–30 cells per animal). Images show maximum intensity projections of 30μm deep stacks. Scale bars represent 100μm.

Article Snippet: The collected brains were postfixed in 4% PFA for 30 min followed by cryoprotection in 30% sucrose; 30-μm-thick sections were cut and stained as previously described ( ; ) with the following primary antibodies: rabbit (rb)-NG2 (1:500, AB5320 Millipore), mouse (m)-GFAP (1:500, G3893 Sigma–Aldrich), and chick-GFP (1:500, GFP-1020 Aves Lab).

Techniques: Migration

Journal: Frontiers in Cell and Developmental Biology

Article Title: NG2-Glia Transiently Overcome Their Homeostatic Network and Contribute to Wound Closure After Brain Injury

doi: 10.3389/fcell.2021.662056

Figure Lengend Snippet: Fast and heterogeneous reaction of NG2-glia after injury. (a) Schematic illustration of the experimental procedure. (b–d) Images of GFP + NG2-glia and oligodendrocytes (white arrows) surrounding a punctate wound injury (PWI; white dashed ellipse) at d0, d2, and d4 after the injury. Blood vessels are labeled with Texas-Red dextran (red). (b’–d’) Examples of cells (higher magnification from b–d ) showing hypertrophy (b’) , the combined reaction of migration and polarization toward the injury ( c’ ; yellow arrow indicates the direction towards the injury) and proliferation (d’) . (e–f) Pie charts represent the heterogeneous reaction of all NG2-glia surrounding the injury site between 0 and 2dpi ( e ; Polarization represents the cells polarizing toward the injury; the classification of the multiple reactions is represented in pie e’ ) and 2 and 4 dpi ( f ; n = 220 cells from 8 animals for d0–d2 and n = 180 cells from 6 animals for d2–d4). Images show maximum intensity projections of 30μm deep stacks. Scale bars represent 100μm in b–d and 25μm in b’–d’ .

Article Snippet: The collected brains were postfixed in 4% PFA for 30 min followed by cryoprotection in 30% sucrose; 30-μm-thick sections were cut and stained as previously described ( ; ) with the following primary antibodies: rabbit (rb)-NG2 (1:500, AB5320 Millipore), mouse (m)-GFAP (1:500, G3893 Sigma–Aldrich), and chick-GFP (1:500, GFP-1020 Aves Lab).

Techniques: Labeling, Migration

Journal: Frontiers in Cell and Developmental Biology

Article Title: NG2-Glia Transiently Overcome Their Homeostatic Network and Contribute to Wound Closure After Brain Injury

doi: 10.3389/fcell.2021.662056

Figure Lengend Snippet: The degree of NG2-glia reaction depends on the size and proximity to the injury. (a,b) Images of the NG2-glia reaction between d0 and d2 after PWI (a,a’) and the bigger stab wound injury (SWI) (b,b’) . (C) NG2-glia show a stronger reaction after SWI compared to PWI (mean + SEM; n = 8 mice for PWI and n = 3 mice for SWI) with a lower percentage of static cells at 2dpi (d; compare to Fig. 1e). (e–g) Cells in closer proximity to the injury show increased reactivity compared to the ones further away from the lesion core at d2 (e,f) . This difference was less pronounced at 4dpi (g, polarization represents cells directed toward the injury; n = 220 cells from 8 animals at d2 and n = 180 cells from 6 animals at d4). Images show maximum intensity projections of 30μm deep stacks. Scale bars represent 100μm.

Article Snippet: The collected brains were postfixed in 4% PFA for 30 min followed by cryoprotection in 30% sucrose; 30-μm-thick sections were cut and stained as previously described ( ; ) with the following primary antibodies: rabbit (rb)-NG2 (1:500, AB5320 Millipore), mouse (m)-GFAP (1:500, G3893 Sigma–Aldrich), and chick-GFP (1:500, GFP-1020 Aves Lab).

Techniques:

Journal: Frontiers in Cell and Developmental Biology

Article Title: NG2-Glia Transiently Overcome Their Homeostatic Network and Contribute to Wound Closure After Brain Injury

doi: 10.3389/fcell.2021.662056

Figure Lengend Snippet: NG2-glia fill the injury core. (a) Images of NG2-glia at d0 (a) and d2 (a’) after PWI. Dotted circle indicates the core of the injury that corresponds to the analyzed area. (b) Graph showing a strong reactivity of these cells for all criteria (except polarization) at d2 and d4 after injury. (c) Pie chart of the heterogeneous reaction between 0 and 2dpi of NG2-glia showing no static cells (Polarization represents cells directed toward the injury; n = 34 cells from 7 animals for d2 and n = 23 cells from 4 animals at d4). Images show maximal projections of 20μm deep stacks. Scale bars represent 100μm. (d–f) Images of 0, 2 and 4 days after SWI showing NG2-glia only filling up the injury core at 4dpi. White ellipse represents the injury site. Images show maximum intensity projections of 30μm deep stacks. Scale bars represent 100μm.

Article Snippet: The collected brains were postfixed in 4% PFA for 30 min followed by cryoprotection in 30% sucrose; 30-μm-thick sections were cut and stained as previously described ( ; ) with the following primary antibodies: rabbit (rb)-NG2 (1:500, AB5320 Millipore), mouse (m)-GFAP (1:500, G3893 Sigma–Aldrich), and chick-GFP (1:500, GFP-1020 Aves Lab).

Techniques:

Journal: Frontiers in Cell and Developmental Biology

Article Title: NG2-Glia Transiently Overcome Their Homeostatic Network and Contribute to Wound Closure After Brain Injury

doi: 10.3389/fcell.2021.662056

Figure Lengend Snippet: Depletion of NG2-glia after injury leads to impaired wound closure. (a) Confocal images of NG2 + cells in Esco wt and Esco fl animals at 2 and 7 dpi. (b) NG2 + cells in Esco wt and Esco fl animals at 4dpi. In Esco fl animals, areas with complete absence of NG2-glia can be observed (dashed ellipse). (c) Cell counts of NG2 + cells per mm 2 in Esco wt and Esco fl animals in control, non-lesioned brains and at different timepoints after the lesion. Esco fl mice show a reduced cell number after injury (n = 3 animals for each genotype and timepoint, cell counts are presented as mean + SEM; 1way ANOVA with Tukey post-test: ∗∗∗ indicates significance level of p < 0.0001). (d) Lesion size in the cerebral cortex visualized by the lack of DAPI positive cells in Esco wt and Esco fl animals at different timepoints after the lesion. (e) Size of the lesion in mm 2 at 2, 4, 7 and 14dpi in Esco wt and Esco fl animals. Esco fl animals show a significantly bigger lesion compared to Esco wt control littermates. (n = 3 for Esco wt (2, 7 and 14dpi), n = 4 for Esco wt (4 and 14dpi), n = 5 for Esco fl (2dpi), n = 6 for Esco fl (4 and 7dpi) animals), data are presented as mean±SEM; 1way ANOVA with Tukey post-test: ∗∗ indicates significance level of p < 0.001. Scale bars represent 25μm in (a) inlays, 50μm in (a) and (b) , 100μm.

Article Snippet: The collected brains were postfixed in 4% PFA for 30 min followed by cryoprotection in 30% sucrose; 30-μm-thick sections were cut and stained as previously described ( ; ) with the following primary antibodies: rabbit (rb)-NG2 (1:500, AB5320 Millipore), mouse (m)-GFAP (1:500, G3893 Sigma–Aldrich), and chick-GFP (1:500, GFP-1020 Aves Lab).

Techniques: Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: NG2-Glia Transiently Overcome Their Homeostatic Network and Contribute to Wound Closure After Brain Injury

doi: 10.3389/fcell.2021.662056

Figure Lengend Snippet: Model scheme of the reaction of NG2-glia at different timepoints after injury.

Article Snippet: The collected brains were postfixed in 4% PFA for 30 min followed by cryoprotection in 30% sucrose; 30-μm-thick sections were cut and stained as previously described ( ; ) with the following primary antibodies: rabbit (rb)-NG2 (1:500, AB5320 Millipore), mouse (m)-GFAP (1:500, G3893 Sigma–Aldrich), and chick-GFP (1:500, GFP-1020 Aves Lab).

Techniques:

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Frizzled 1 and Wnt1 as new potential therapeutic targets in the traumatically injured spinal cord

doi: 10.1007/s00018-019-03427-4

Figure Lengend Snippet: Primary and secondary antibodies used for immunohistochemistry

Article Snippet: , , Rb anti-NG2 , AB5320, Millipore , 1:250.

Techniques: Immunohistochemical staining, Immunohistochemistry, Plasmid Preparation, Fluorescence, Marker