mouse monoclonal antibody tu 20  (Novus Biologicals)

Journal: Cell Death Discovery

Article Title: Protein expression of nucleolar protein 12 in the retina and its implication in protection of retina from UV irradiation damage

doi: 10.1038/s41420-024-01902-x

Figure Lengend Snippet: A Double immunofluorescence staining for analyzing subcellular localization of NOL12 in the cells of rat retinal tissue, showing the colocalization of NOL12 with the RGCs marker BRN3B (a1) and the dendrite marker MAP2 (a2), but not with the axonal terminal marker GAP43 (a3). B Double immunofluorescence staining of NOL12 and the nucleolar marker fibrillarin in WERI-Rb1 cells showing that NOL12 is primarily distributed in the nucleolus, with a low level of cytoplasmic distribution (arrowheads). High-power images of the boxed areas in B are shown in b1–b4. A rabbit polyclonal antibody against NOL12 was used in immunofluorescence staining in a1, a3, and B , while a mouse monoclonal antibody against NOL12 was used in immunofluorescence staining in a2. NOL12 was labeled by RRX (red), BRN3B, GAP43, MAP2, and fibrillarin were labeled by FITC (green), and nuclei were counterstained with Hoechst 33258 (blue). RGCs, retinal ganglion cells. Scale bar: 20 μm in A and B , 5 μm in b1.

Article Snippet: Sigma (MO, USA) supplied us with the mouse monoclonal antibody against γ-tubulin (T6557), while Cell Signaling Technology (MA, USA) provided the rabbit monoclonal antibody against cleaved Caspase-3 (9661).

Techniques: Double Immunofluorescence Staining, Marker, Immunofluorescence, Staining, Labeling

Journal: Cell Death Discovery

Article Title: Protein expression of nucleolar protein 12 in the retina and its implication in protection of retina from UV irradiation damage

doi: 10.1038/s41420-024-01902-x

Figure Lengend Snippet: A Double immunofluorescence staining of NOL12 and ATR in WERI-Rb1 cells showing that NOL12 is well colocalized with ATR in WERI-Rb1 cells, predominantly in the nucleoli (arrowheads) and to a lesser extent in the cytoplasm (arrows). High-power images of the boxed areas in ( A ) are shown in a1–a8. B The interactions between NOL12, ATR, and its substrate RPA in WERI-Rb1 cells were detected by co-immunoprecipitation assays. C Double immunofluorescence staining of NOL12 and ATR in adult rat retinas showing that NOL12 is well colocalized with ATR in the cytoplasm of RGCs. High-power images of the boxed areas in ( C ) are shown in c1–c4. D The interactions between NOL12, ATR, and its substrate RPA in adult rat retinas were detected by co-immunoprecipitation assays. Rabbit polyclonal antibody against NOL12 and mouse monoclonal antibody against ATR was utilized in immunofluorescence staining ( A , C ). Rabbit polyclonal antibodies against NOL12 and ATR were utilized in Western blot ( B , D ). NOL12 is labeled by RRX (red), ATR by FITC (green), and nuclei are counterstained with Hoechst 33258 (blue) ( A , C ). Scale bars: 5 μm in A and 10 μm in C .

Article Snippet: Sigma (MO, USA) supplied us with the mouse monoclonal antibody against γ-tubulin (T6557), while Cell Signaling Technology (MA, USA) provided the rabbit monoclonal antibody against cleaved Caspase-3 (9661).

Techniques: Double Immunofluorescence Staining, Immunoprecipitation, Immunofluorescence, Staining, Western Blot, Labeling

Journal: Neurobiology of disease

Article Title: GPR3 expression in retinal ganglion cells contributes to neuron survival and accelerates axonal regeneration after optic nerve crush in mice.

doi: 10.1016/j.nbd.2022.105811

Figure Lengend Snippet: Fig. 1. G protein-coupled receptor 3 (GPR3) expression in the mouse retina. (A–B) GPR3 expression was investigated using PA-tagged GPR3 knockin mice (PA-GPR3 mice). Retinal sections were stained with an anti-PA antibody. (A) A representative coronal section stained with an anti-PA antibody is shown. (B) A magnified image of the GPR3-positive area in the retina (enclosed in a dotted square in A) is shown. (C–N) Retinal sections from PA-GPR3 mice were double stained with an anti-PA antibody and an anti-βIII Tubulin antibody (C–E), anti-Brn3a antibody (F–H), anti-PAX6 antibody (I–K), or anti-GLAST antibody (L–N). The insets show magnified images of the dotted squares. Scale bar = 20 μm.

Article Snippet: After blocking with 3% NGS, the sections were then incubated overnight at 4 ◦C in the following primary antibodies diluted at 1:200 in PBS unless otherwise indicated: anti- βIII Tubulin Monoclonal Antibody (clone TuJ1) (#4466, Cell Signaling Technology, Danvers, MA, USA), anti-GLAST (EAAT1) rabbit monoclonal antibody (#5684, Cell Signaling Technology), anti-Brn3a rabbit polyclonal antibody (ab245230, Abcam, Cambridge, UK), anti-PAX6 rabbit polyclonal antibody (ab5790, Abcam), anti-cAMP rabbit polyclonal antibody (20–198, Sigma-Aldrich), and anti-pERK1/2 antibody (#4370S, Cell Signaling Technology).

Techniques: Expressing, Knock-In, Staining

Journal: Neurobiology of disease

Article Title: GPR3 expression in retinal ganglion cells contributes to neuron survival and accelerates axonal regeneration after optic nerve crush in mice.

doi: 10.1016/j.nbd.2022.105811

Figure Lengend Snippet: Fig. 4. G protein-coupled receptor 3 (GPR3) participates in both neurite outgrowth and neuronal survival in mouse primary cultured RGCs. (A–B) Primary cultured RGCs were prepared from P4 neonatal mice and plated onto poly-L-lysine-coated plates. (A) Retinal neurons were then fixed at 0, 1, 2, 4, and 7 DIV and stained with an anti-βIII Tubulin antibody (green). Representative images from retinal neurons captured by confocal microscopy at different time points are shown. Scale bar = 100 μm. (B) Intrinsic GPR3 expression following culture was evaluated using real-time RT-PCR. The graph shows the results from three independent replicates. Values indicate the mean ± SEM in each group. (C–D) RGCs were transfected with either control siRNA + pMAX-EGFP or GPR3 siRNA + pMAX-EGFP plasmid vectors. Twenty-four and 48 h after transfection, neurons were fixed, and the length of the longest neurite of each GFP-positive neuron was measured. (C) Representative images from mouse retinal neurons in each condition. Scale bar = 10 μm. (D) Neurite lengths were compared between control siRNA- and GPR3siRNA-treated neurons. The graph shows the results from four independent replicates. Values indicate the mean ± SEM in each group. (E–F) RGCs were transfected with either pc-mAGFL (mock) or pc-GPR3mAGFL plasmid vectors. Twenty-four and 48 h after transfection, neurons were fixed, and the length of the longest neurite of each GFP-positive neuron was measured. (E) Representative images from mouse retinal neurons in each condition. Scale bar = 10 μm. (F) Neurite lengths were compared between mock and GPR3-expressing RGCs. The graph shows the results from three independent replicates. Values indicate the mean ± SEM in each group. (G–H) The survival of neurons in culture was assessed by DAPI staining. (G) Representative images from control siRNA- and GPR3 siRNA-transfected RGCs at various time points. Scale bar = 10 μm. (H) The ratio of surviving neurons to the total number of DAPI-stained cells at various time points is shown. All graphs in this figure show the results from four independent replicates. Values indicate the mean ± SEM. Statistical significance was evaluated using one-way ANOVA followed by a post hoc Dunnett test (B) or Bonferroni test (D, F, H). *, p < 0.05; **, p < 0.01; ***, p < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: After blocking with 3% NGS, the sections were then incubated overnight at 4 ◦C in the following primary antibodies diluted at 1:200 in PBS unless otherwise indicated: anti- βIII Tubulin Monoclonal Antibody (clone TuJ1) (#4466, Cell Signaling Technology, Danvers, MA, USA), anti-GLAST (EAAT1) rabbit monoclonal antibody (#5684, Cell Signaling Technology), anti-Brn3a rabbit polyclonal antibody (ab245230, Abcam, Cambridge, UK), anti-PAX6 rabbit polyclonal antibody (ab5790, Abcam), anti-cAMP rabbit polyclonal antibody (20–198, Sigma-Aldrich), and anti-pERK1/2 antibody (#4370S, Cell Signaling Technology).

Techniques: Cell Culture, Staining, Confocal Microscopy, Expressing, Quantitative RT-PCR, Transfection, Control, Plasmid Preparation

Journal: Neurobiology of disease

Article Title: GPR3 expression in retinal ganglion cells contributes to neuron survival and accelerates axonal regeneration after optic nerve crush in mice.

doi: 10.1016/j.nbd.2022.105811

Figure Lengend Snippet: Fig. 6. Adeno-associated virus (AAV)-mediated G protein-coupled receptor 3 (GPR3) gene transduction in RGCs and its effect on downstream pathways in mice. (A) rAAV-mAGFL (rAAV-mock) or rAAV-GPR3mAGFL (rAAV-GPR3) was administered in the vitreous of the mouse retina. Twelve days after inoculation, the retina was fixed, and fluorescent GFP was evaluated by confocal micro scopy. Representative images from rAAV-mock or rAAV-GPR3 transfected ret inas stained with an anti-βIII Tubulin antibody are shown. The right insets show magnified images of the dotted squares. Scale bar = 25 μm. (B–C) Intracellular cAMP elevation and ERK1/2 phosphorylation were evaluated in rAAV-mock and rAAV-GPR3 transfected RGCs. Retinal sections from each condition were stained with an anti-cAMP antibody (B) and an anti-pERK1/2 antibody (C) 2 weeks after AAV transfection. The insets show magnified images of the dotted squares. Scale bar = 50 μm.

Article Snippet: After blocking with 3% NGS, the sections were then incubated overnight at 4 ◦C in the following primary antibodies diluted at 1:200 in PBS unless otherwise indicated: anti- βIII Tubulin Monoclonal Antibody (clone TuJ1) (#4466, Cell Signaling Technology, Danvers, MA, USA), anti-GLAST (EAAT1) rabbit monoclonal antibody (#5684, Cell Signaling Technology), anti-Brn3a rabbit polyclonal antibody (ab245230, Abcam, Cambridge, UK), anti-PAX6 rabbit polyclonal antibody (ab5790, Abcam), anti-cAMP rabbit polyclonal antibody (20–198, Sigma-Aldrich), and anti-pERK1/2 antibody (#4370S, Cell Signaling Technology).

Techniques: Virus, Transduction, Transfection, Staining, Phospho-proteomics