mouse syt1, by Synaptic Systems - Product details

1

Quantifying Viral Protein and Synaptic Marker Overlap

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Vibratome sections (100 μm thick) from virus-injected animals were immunolabeled with antibodies against mCherry, which was expressed from the virus, as well as antibodies against the mossy fiber marker, ZnT3. CellProfiler (Version 2.1.0) software was used to quantify the degree of overlap between mCherry and ZnT3. The average amount of overlap was 70%.
Primary antibodies: rat BrdU (AbD Serotec, MCA2060GA, 1:500), rabbit GFP (Invitrogen, A11122, 1:300), rabbit Complexin-1 (Proteintech, 10246–2–AP, 1:300), mouse GFAP (G6171, 1:500), mouse Nsf-1 (MA1–12435, 1:300) and rabbit Syn3 (OSS00018W, 1:300) from Thermo Scientific, guinea pig Iba1 (234 004, 1:500), mouse Syt1 (105 011, 1:300), guinea pig ZnT3 (197 004, 1:1000) from Synaptic Systems.
Secondary antibodies. Antibodies (all obtained from Jackson Laboratory, donkey anti-guinea pig IgG (H+L) ML, #706-225-148 and 706-175-148; donkey anti-mouse IgG (H+L) ML, #715-225-151, 715-165-151, and 715-175-151; donkey anti-rabbit IgG (H+L) ML, #711-225-152, 711-165-152, and 711-175-152 were diluted 1:400. Confocal scans were carried out using a LSM 510 Imager Z.1 microscope (Zeiss) using Plan-Apochromat 63X/1.4 and EC Plan-Neofluar 40X/1.30 oil immersion objective lenses and four excitation laser lines. Tile scans were performed using a LSM 710 confocal microscope (Zeiss) with a Plan-Apochromat 20X/0.8 objective.

Siegert S., Seo J., Kwon E.J., Rudenko A., Cho S., Wang W., Flood Z., Martorell A.J., Ericsson M., Mungenast A.E, & Tsai L.H. (2015). The schizophrenia risk gene product miR-137 alters presynaptic plasticity. Nature neuroscience, 18(7), 1008-1016.

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2

Western Blot Analysis of Synaptic Proteins

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Protein was transferred to Immobilion PVDF transfer membrane (Millipore) in transfer buffer (25 mM Tris, 192 mM glycine, 20% (v/v) methanol) for 1 hr at 100 V constant, 350 mA. The membrane was blocked for at least 1 h in 5% (wt/vol) non-fat dry milk in Tris-buffered saline (25 mM Tris, 150 mM NaCl, 2 mM KCl, pH 8.0, 15 mM NaCl, 10 mM Tris, pH 8.0). Primary rabbit Complexin-1 (Proteintech, 10246–2–AP), mouse Nsf-1 (MA1–12435) and rabbit Syn3 (OSS00018W, 1:300) from Thermo Scientific, mouse Syt1 (Synaptic Systems, 105 011, mouse β-actin (Sigma Aldrich, A5316) were added 1:2,000 in blocking solution and incubated for 2–3 days on a shaker at 4 °C. The membrane was washed 3× for 30 min in Tris-buffered saline and incubated with secondary antibody (1:10,000, Li-Cor: IRDye 680 RD donkey anti-mouse IgG (H+L), #925-68072; IRDye 680RD donkey anti-rabbit IgG (H+L), #925-68073; IRDye 800CW donkey anti-mouse IgG (H+L), #925-32212; IRDye 800 CW donkey anti-rabbit IgG (H+L), #925-32213. Detection was performed using an Odyssey machine (Li-Cor). Image Studio 2.0 software was used for image analysis. The membrane was stripped with NewBlot PVDF 5x stripping buffer (Li-Cor) according to manufacturer’s protocol. We normalized and corrected for uneven loading using the intensity of the internal control band, beta-Actin.

Siegert S., Seo J., Kwon E.J., Rudenko A., Cho S., Wang W., Flood Z., Martorell A.J., Ericsson M., Mungenast A.E, & Tsai L.H. (2015). The schizophrenia risk gene product miR-137 alters presynaptic plasticity. Nature neuroscience, 18(7), 1008-1016.

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3

Immunofluorescent Labeling of Neuronal Markers

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Primary antibodies used were: chick and rabbit EGFP (1:4,000 dilution; Abcam), mouse GAD65 (1:1,000), guinea pig (1:800 for uptake) and mouse syt1 (1:150 for uptake), rabbit alpha tubulin (1:5,000), mouse syb2 (1:1,000 dilution; Synaptic Systems), chick and mouse MAP2 (1:4,000 dilution; Millipore), sheep NGF, mouse reelin, guinea pig TRPV1 C-T (1:1,000, Millipore), mouse TRPV1 N-T (1:500; Neuromab), rabbit TRPV2, TRPV3, TRPV4 (1:500; Alomone), rabbit mGluR7 (1:500; Upstate), mouse PV25 (1:2,000 dilution; Swant), rabbit Somatostatin-14 (1:2,000 dilution; Bachem), rat Somatostatin-14 (1:1,000 dilution; Thermo Fisher), rabbit VIP (1:500 dilution; Abcam), mouse BDNF (1:500; Developmental Studies Hybridoma Bank; Yves Barde monoclonal #9). Secondary antibodies used were: guinea pig, mouse and rabbit HRP (1:5,000 dilution; BioRad) and chick, rabbit, guinea pig, mouse, rat and sheep Alexa 405, 488, 546 and 647 (1:2,000 dilution; Abcam/Invitrogen). Neurotrace was from Thermo Fisher.
Capsaicin and SB-366791 were purchased from Sigma-Aldrich. Methyllycaconitine (MLA) was from Abcam and dihydro-beta-erythroidine (DHBE), mecamylamine (MEC) picrotoxin, TTX, APV and CNQX were from Tocris. Recombinant Human TrkA Fc and TrkB Fc Chimera Proteins were from R & D Systems. All other reagents were from Carl Roth.

Hurtado-Zavala J.I., Ramachandran B., Ahmed S., Halder R., Bolleyer C., Awasthi A., Stahlberg M.A., Wagener R.J., Anderson K., Drenan R.M., Lester H.A., Miwa J.M., Staiger J.F., Fischer A, & Dean C. (2017). TRPV1 regulates excitatory innervation of OLM neurons in the hippocampus. Nature Communications, 8, 15878.

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4

Quantifying Viral Protein and Synaptic Marker Overlap

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Vibratome sections (100 μm thick) from virus-injected animals were immunolabeled with antibodies against mCherry, which was expressed from the virus, as well as antibodies against the mossy fiber marker, ZnT3. CellProfiler (Version 2.1.0) software was used to quantify the degree of overlap between mCherry and ZnT3. The average amount of overlap was 70%.
Primary antibodies: rat BrdU (AbD Serotec, MCA2060GA, 1:500), rabbit GFP (Invitrogen, A11122, 1:300), rabbit Complexin-1 (Proteintech, 10246–2–AP, 1:300), mouse GFAP (G6171, 1:500), mouse Nsf-1 (MA1–12435, 1:300) and rabbit Syn3 (OSS00018W, 1:300) from Thermo Scientific, guinea pig Iba1 (234 004, 1:500), mouse Syt1 (105 011, 1:300), guinea pig ZnT3 (197 004, 1:1000) from Synaptic Systems.
Secondary antibodies. Antibodies (all obtained from Jackson Laboratory, donkey anti-guinea pig IgG (H+L) ML, #706-225-148 and 706-175-148; donkey anti-mouse IgG (H+L) ML, #715-225-151, 715-165-151, and 715-175-151; donkey anti-rabbit IgG (H+L) ML, #711-225-152, 711-165-152, and 711-175-152 were diluted 1:400. Confocal scans were carried out using a LSM 510 Imager Z.1 microscope (Zeiss) using Plan-Apochromat 63X/1.4 and EC Plan-Neofluar 40X/1.30 oil immersion objective lenses and four excitation laser lines. Tile scans were performed using a LSM 710 confocal microscope (Zeiss) with a Plan-Apochromat 20X/0.8 objective.

Siegert S., Seo J., Kwon E.J., Rudenko A., Cho S., Wang W., Flood Z., Martorell A.J., Ericsson M., Mungenast A.E, & Tsai L.H. (2015). The schizophrenia risk gene product miR-137 alters presynaptic plasticity. Nature neuroscience, 18(7), 1008-1016.

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5

Western Blot Analysis of Synaptic Proteins

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Protein was transferred to Immobilion PVDF transfer membrane (Millipore) in transfer buffer (25 mM Tris, 192 mM glycine, 20% (v/v) methanol) for 1 hr at 100 V constant, 350 mA. The membrane was blocked for at least 1 h in 5% (wt/vol) non-fat dry milk in Tris-buffered saline (25 mM Tris, 150 mM NaCl, 2 mM KCl, pH 8.0, 15 mM NaCl, 10 mM Tris, pH 8.0). Primary rabbit Complexin-1 (Proteintech, 10246–2–AP), mouse Nsf-1 (MA1–12435) and rabbit Syn3 (OSS00018W, 1:300) from Thermo Scientific, mouse Syt1 (Synaptic Systems, 105 011, mouse β-actin (Sigma Aldrich, A5316) were added 1:2,000 in blocking solution and incubated for 2–3 days on a shaker at 4 °C. The membrane was washed 3× for 30 min in Tris-buffered saline and incubated with secondary antibody (1:10,000, Li-Cor: IRDye 680 RD donkey anti-mouse IgG (H+L), #925-68072; IRDye 680RD donkey anti-rabbit IgG (H+L), #925-68073; IRDye 800CW donkey anti-mouse IgG (H+L), #925-32212; IRDye 800 CW donkey anti-rabbit IgG (H+L), #925-32213. Detection was performed using an Odyssey machine (Li-Cor). Image Studio 2.0 software was used for image analysis. The membrane was stripped with NewBlot PVDF 5x stripping buffer (Li-Cor) according to manufacturer’s protocol. We normalized and corrected for uneven loading using the intensity of the internal control band, beta-Actin.

Siegert S., Seo J., Kwon E.J., Rudenko A., Cho S., Wang W., Flood Z., Martorell A.J., Ericsson M., Mungenast A.E, & Tsai L.H. (2015). The schizophrenia risk gene product miR-137 alters presynaptic plasticity. Nature neuroscience, 18(7), 1008-1016.

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Mouse syt1

Lab products found in correlation

5 protocols using mouse syt1

Quantifying Viral Protein and Synaptic Marker Overlap

Western Blot Analysis of Synaptic Proteins

Immunofluorescent Labeling of Neuronal Markers

Quantifying Viral Protein and Synaptic Marker Overlap

Western Blot Analysis of Synaptic Proteins

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