Blocking Assay:Article Title: RIM-BP2 regulates Ca 2+ channel abundance and neurotransmitter release at hippocampal mossy fiber terminals
Article Snippet: .. After blocking with PBS containing 0.3% BSA at room temperature, sections were incubated with primary antibodies diluted in PBS containing 0.3% BSA: anti-VGLUT1 (1:2000, Synaptic Systems, guinea pig polyclonal, RRID: AB_887878 ), anti-PSD-95 (1:50, NeuroMab, mouse monoclonal IgG2a, clone K28/43, TC Supernatant, RRID: AB_2877189 ), anti-Munc13-1 (1:1000, mouse monoclonal IgG1, clone 11B-10G, ), and anti-Cav2.1 (1:400, Synaptic Systems, rabbit polyclonal, RRID: AB_2619841 ) or anti-Cav2.2 (1:400, Synaptic Systems, mouse monoclonal IgG2b, clone 163E3, RRID: AB_2619843 ) or anti-RIM-BP2 (1:200, mouse monoclonal IgG2b, clone 8-4G, ) for 3 hr at room temperature. .. Afterward, sections were washed and incubated with fluorescence dye-labeled secondary antibodies: DyLight 405 Anti-Guinea Pig IgG (Jackson ImmunoResearch, RRID: AB_2340470 ), Alexa Fluor 488 (Thermo Fisher Scientific)-labeled Anti-Mouse IgG2a (Jackson ImmunoResearch, RRID: AB_2338462 ), STAR635P (Abberior)-labeled Anti-Mouse IgG1 (Jackson ImmunoResearch, RRID: AB_2338461 ), and Alexa Fluor 594 (Thermo Fisher Scientific)-labeled Anti-Rabbit IgG (Jackson ImmunoResearch, RRID: AB_2340585 ) or Anti-Mouse IgG2b (Jackson ImmunoResearch, RRID: AB_2338463 ) for 1 hr at room temperature.
Article Title: RIM-BP2 regulates Ca2+ channel abundance and neurotransmitter release at hippocampal mossy fiber terminals
Article Snippet: .. After blocking with PBS containing 0.3% BSA at room temperature, sections were incubated with primary antibodies diluted in PBS containing 0.3% BSA: anti- VGLUT1 (1:2000, Synaptic Systems, guinea pig polyclonal, RRID:AB_887878), anti- PSD- 95 (1:50, NeuroMab, mouse monoclonal IgG2a, clone K28/43, TC Supernatant, RRID:AB_2877189), anti- Munc13- 1 (1:1000, mouse monoclonal IgG1, clone 11B- 10G, Sakamoto et al., 2018), and anti- Cav2.1 (1:400, Synaptic Systems, rabbit polyclonal, RRID:AB_2619841) or anti- Cav2.2 (1:400, Synaptic Systems, mouse monoclonal IgG2b, clone 163E3, RRID:AB_2619843) or anti- RIM- BP2 (1:200, mouse monoclonal IgG2b, clone 8- 4G, Sakamoto et al., 2022) for 3 hr at room temperature. .. Afterward, sections were washed and incubated with fluorescence dye- labeled secondary antibodies: DyLight 405 Anti- Guinea Pig IgG (Jackson ImmunoResearch, RRID:AB_2340470), Alexa Fluor 488 (Thermo Fisher Scientific)- labeled Anti- Mouse IgG2a (Jackson ImmunoResearch, RRID:AB_2338462), STAR635P (Abberior)- labeled Anti- Mouse IgG1 (Jackson ImmunoResearch, RRID:AB_2338461), and Alexa Fluor 594 (Thermo Fisher Scientific)- labeled AntiRabbit IgG (Jackson ImmunoResearch, RRID:AB_2340585) or Anti- Mouse IgG2b (Jackson ImmunoResearch, RRID:AB_2338463) for 1 hr at room temperature.
Article Title: Cypin regulates K63-linked polyubiquitination to shape synaptic content
Article Snippet: .. Membranes were then incubated with primary antibodies diluted in blocking solution at 1:500 (anti-Ub, Cytoskeleton, catalog no. AUB01), 1:1000 (anti-HA, Cell Signaling, catalog no. 3724; anti-PSD-95, Neuromab, catalog no. 75-028; anti-K63-polyUb, Cell Signaling, catalog no. 5621; anti-K48-Ub, Cell Signaling, catalog no. 8081; anti–β-actin, Cell Signaling, catalog no. 3700; anti-GluN2A, Cell Signaling, catalog no. 4205; anti-GluN2B, Cell Signaling, catalog no. 4207; anti-GluR1, Cell Signaling, catalog no. 13185; anti-UBE4A, Santa Cruz, catalog no. sc-365904; anti-PSMC2, Santa Cruz, catalog no. sc-166972; anti-UBE2J1, Santa Cruz, catalog no. sc-377002; anti-proteasome 20 S α1, 2, 3, 5, 6, and 7 subunits, Enzo, catalog no. BML-PW8195; anti-Rpt5, Enzo, catalog no. BML-PW8770; anti-Rpt6, Enzo, catalog no. BML-PW9265; anti-cypin, Firestein lab BF6 clone) overnight at 4°C. .. Membranes were washed three times with TBS-T and incubated with respective horseradish peroxidase–conjugated secondary antibodies (1:2500; Rockland) or fluorescently labeled secondary antibodies (1:2500; LI-COR) in blocking solution for 1 hour at room temperature.
Incubation:Article Title: RIM-BP2 regulates Ca 2+ channel abundance and neurotransmitter release at hippocampal mossy fiber terminals
Article Snippet: .. After blocking with PBS containing 0.3% BSA at room temperature, sections were incubated with primary antibodies diluted in PBS containing 0.3% BSA: anti-VGLUT1 (1:2000, Synaptic Systems, guinea pig polyclonal, RRID: AB_887878 ), anti-PSD-95 (1:50, NeuroMab, mouse monoclonal IgG2a, clone K28/43, TC Supernatant, RRID: AB_2877189 ), anti-Munc13-1 (1:1000, mouse monoclonal IgG1, clone 11B-10G, ), and anti-Cav2.1 (1:400, Synaptic Systems, rabbit polyclonal, RRID: AB_2619841 ) or anti-Cav2.2 (1:400, Synaptic Systems, mouse monoclonal IgG2b, clone 163E3, RRID: AB_2619843 ) or anti-RIM-BP2 (1:200, mouse monoclonal IgG2b, clone 8-4G, ) for 3 hr at room temperature. .. Afterward, sections were washed and incubated with fluorescence dye-labeled secondary antibodies: DyLight 405 Anti-Guinea Pig IgG (Jackson ImmunoResearch, RRID: AB_2340470 ), Alexa Fluor 488 (Thermo Fisher Scientific)-labeled Anti-Mouse IgG2a (Jackson ImmunoResearch, RRID: AB_2338462 ), STAR635P (Abberior)-labeled Anti-Mouse IgG1 (Jackson ImmunoResearch, RRID: AB_2338461 ), and Alexa Fluor 594 (Thermo Fisher Scientific)-labeled Anti-Rabbit IgG (Jackson ImmunoResearch, RRID: AB_2340585 ) or Anti-Mouse IgG2b (Jackson ImmunoResearch, RRID: AB_2338463 ) for 1 hr at room temperature.
Article Title: RIM-BP2 regulates Ca2+ channel abundance and neurotransmitter release at hippocampal mossy fiber terminals
Article Snippet: .. After blocking with PBS containing 0.3% BSA at room temperature, sections were incubated with primary antibodies diluted in PBS containing 0.3% BSA: anti- VGLUT1 (1:2000, Synaptic Systems, guinea pig polyclonal, RRID:AB_887878), anti- PSD- 95 (1:50, NeuroMab, mouse monoclonal IgG2a, clone K28/43, TC Supernatant, RRID:AB_2877189), anti- Munc13- 1 (1:1000, mouse monoclonal IgG1, clone 11B- 10G, Sakamoto et al., 2018), and anti- Cav2.1 (1:400, Synaptic Systems, rabbit polyclonal, RRID:AB_2619841) or anti- Cav2.2 (1:400, Synaptic Systems, mouse monoclonal IgG2b, clone 163E3, RRID:AB_2619843) or anti- RIM- BP2 (1:200, mouse monoclonal IgG2b, clone 8- 4G, Sakamoto et al., 2022) for 3 hr at room temperature. .. Afterward, sections were washed and incubated with fluorescence dye- labeled secondary antibodies: DyLight 405 Anti- Guinea Pig IgG (Jackson ImmunoResearch, RRID:AB_2340470), Alexa Fluor 488 (Thermo Fisher Scientific)- labeled Anti- Mouse IgG2a (Jackson ImmunoResearch, RRID:AB_2338462), STAR635P (Abberior)- labeled Anti- Mouse IgG1 (Jackson ImmunoResearch, RRID:AB_2338461), and Alexa Fluor 594 (Thermo Fisher Scientific)- labeled AntiRabbit IgG (Jackson ImmunoResearch, RRID:AB_2340585) or Anti- Mouse IgG2b (Jackson ImmunoResearch, RRID:AB_2338463) for 1 hr at room temperature.
Article Title: Cypin regulates K63-linked polyubiquitination to shape synaptic content
Article Snippet: .. Membranes were then incubated with primary antibodies diluted in blocking solution at 1:500 (anti-Ub, Cytoskeleton, catalog no. AUB01), 1:1000 (anti-HA, Cell Signaling, catalog no. 3724; anti-PSD-95, Neuromab, catalog no. 75-028; anti-K63-polyUb, Cell Signaling, catalog no. 5621; anti-K48-Ub, Cell Signaling, catalog no. 8081; anti–β-actin, Cell Signaling, catalog no. 3700; anti-GluN2A, Cell Signaling, catalog no. 4205; anti-GluN2B, Cell Signaling, catalog no. 4207; anti-GluR1, Cell Signaling, catalog no. 13185; anti-UBE4A, Santa Cruz, catalog no. sc-365904; anti-PSMC2, Santa Cruz, catalog no. sc-166972; anti-UBE2J1, Santa Cruz, catalog no. sc-377002; anti-proteasome 20 S α1, 2, 3, 5, 6, and 7 subunits, Enzo, catalog no. BML-PW8195; anti-Rpt5, Enzo, catalog no. BML-PW8770; anti-Rpt6, Enzo, catalog no. BML-PW9265; anti-cypin, Firestein lab BF6 clone) overnight at 4°C. .. Membranes were washed three times with TBS-T and incubated with respective horseradish peroxidase–conjugated secondary antibodies (1:2500; Rockland) or fluorescently labeled secondary antibodies (1:2500; LI-COR) in blocking solution for 1 hour at room temperature.
Staining:Article Title: ATLAS: a rationally designed anterograde transsynaptic tracer
Article Snippet: .. Neurons were stained using: anti-GFP, 1:10,000, Aves GFP-1020; anti-MYC, 1:1,000, Novus Biologicals NB600-334; anti-Map2, 1:1,000, Abcam ab5392; anti-clathrin, 1:1,000, GeneTex GTX80218; anti-mCherry, 1:1,000, Rockland M11217 ; anti-RFP, 1:1,000, Invitrogen MA5-15257; anti-GluA1, 1:1,000, EMD Millipore ABN241; anti-PSD-95, 1:3,000, NeuroMab 75-348; anti-HA, 1:1,000, Cell Signaling 3724S; anti-Cre, 1:1,000, EMD Millipore MAB3120; Alexa Fluor 488 goat anti-chicken, 1:1,000, Invitrogen A-11006; Alexa Fluor 594 goat anti-rabbit, 1:1,000, Invitrogen A-11012; Alexa Fluor 594 goat anti-mouse, 1:1,000, Invitrogen A-11005; Alexa Fluor 647 goat anti-mouse, 1:1,000, Invitrogen A-21235; Alexa Fluor 647 goat anti-rabbit, 1:1,000, Invitrogen A-21244; JF549 halotag ligand, 1:1,000, Janelia Farm; JF647 halotag ligand, 1:1,000, Janelia Farm; FluoTag-X2 anti-alfa 647, 1:1,000, NanoTag biotechnologies N1502 AF647. ..
Article Title: ATLAS: a rationally designed anterograde transsynaptic tracer.
Article Snippet: .. Neurons were stained using: anti-GFP, 1:10,000, Aves GFP-1020; anti-MYC, 1:1,000, Novus Biologicals NB600-334; anti-Map2, 1:1,000, Abcam ab5392; anti-clathrin, 1:1,000, GeneTex GTX80218; anti-mCherry, 1:1,000, Rockland M11217; anti-RFP, 1:1,000, Invitrogen MA5-15257; anti-GluA1, 1:1,000, EMD Millipore ABN241; anti-PSD-95, 1:3,000, NeuroMab 75-348; anti-HA, 1:1,000, Cell Signaling 3724S; anti-Cre, 1:1,000, EMD Millipore MAB3120; Alexa Fluor 488 goat anti-chicken, 1:1,000, Invitrogen A-11006; Alexa Fluor 594 goat anti-rabbit, 1:1,000, Invitrogen A-11012; Alexa Fluor 594 goat anti-mouse, 1:1,000, Invitrogen A-11005; Alexa Fluor 647 goat anti-mouse, 1:1,000, Invitrogen A-21235; Alexa Fluor 647 goat anti-rabbit, 1:1,000, Invitrogen A-21244; JF549 halotag ligand, 1:1,000, Janelia Farm; JF647 halotag ligand, 1:1,000, Janelia Farm; FluoTag-X2 anti-alfa 647, 1:1,000, NanoTag biotechnologies N1502 AF647. ..
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