Mouse primary astrocyte and E14 primary neuronal cell lysates were fractionated using SDS-PAGE and transferred onto a PVDF (Millipore, Bedford, MA, USA) membrane using a transfer apparatus according to the manufacturer’s protocols (Bio-Rad Laboratories, Inc., Hercules, CA, USA). After incubation with 5% non-fat milk in TBST (10 mM Tris (pH 8.0), 150 mM NaCl, and 0.05% Tween 20) for 1 h, the membranes were incubated with anti-Sp1, anti-NeuN, anti-GFAP (Millipore), anti-MAP2, anti-SNAP25 (Abcam), anti-N-cadherin (GeneTex, CA, USA), and anti-actin (Sigma) antibodies at 4 °C overnight. The membranes were then washed with TBST and incubated with secondary antibodies (Millipore) at room temperature for 1 h blots. After washing with TBST, blots were developed using the ECL system.
Anti snap25
Manufactured by Abcam
Sourced in United States
Anti-SNAP25 is a primary antibody used to detect the presence and distribution of SNAP25 (Synaptosomal-Associated Protein 25) in biological samples. SNAP25 is a protein involved in the docking and fusion of synaptic vesicles with the presynaptic plasma membrane, a critical process in neurotransmitter release.
Automatically generated - may contain errors
Lab products found in correlation
Anti neun, by Merck Group (4 mentions)
Anti h3k122ac, by Abcam (2 mentions)
Anti h4k12ac, by Abcam (2 mentions)
Anti acss2 t, by Thermo Fisher Scientific (2 mentions)
Anti acss2 cs, by Cell Signaling Technology (2 mentions)
Anti acl, by Proteintech (2 mentions)
Anti kat3a cbp, by Abcam (2 mentions)
Anti map2 c d, by Cell Signaling Technology (2 mentions)
Anti nr4a2, by Santa Cruz Biotechnology (2 mentions)
Anti synaptophysin, by Merck Group (2 mentions)
Anti h4k5ac, by Merck Group (2 mentions)
Anti h4, by Merck Group (2 mentions)
Anti α tubulin, by Merck Group (2 mentions)
Anti h3k27ac, by Abcam (2 mentions)
Anti h3k9ac, by Abcam (2 mentions)
Anti h3, by Abcam (2 mentions)
Anti gapdh, by Biosynth (2 mentions)
Benzonase nuclease, by Merck Group (2 mentions)
Protease and phosphatase inhibitor cocktail, by Merck Group (2 mentions)
Anti gfap, by BD (2 mentions)
11 protocols using anti snap25
1
Immunoblotting of Mouse Primary Astrocytes and Neurons
2
Western Blot Analysis of Synaptic Proteins
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Total protein samples for western blot analysis were extracted from cultured NRNs or the left ventricle and hippocampi of the mice after they were anaesthetized with sodium pentobarbital (100 mg/kg, i.p.). Mouse death was then confirmed by exsanguination according to a previously described method [31 (link)]. Hippocampi for primary cell culture were collected from neonatal Sprague-Dawley (SD) rats after the administration of 20% isoflurane and confirmation of death by cervical dislocation. Anti-SNAP-25 (1:1000, ab5666, Abcam, MA, USA), anti-VAMP-2 (1:10000, 104,211, Synaptic Systems, Gottingen, Germany) and anti-Syntaxin-1A (1:5000, 100,111, Synaptic Systems, Gottingen, Germany), anti-Munc-18 (1:1000, 116,002, Synaptic Systems, Gottingen, Germany), anti-CD63 (1:1000, ab193349, Abcam, MA, USA),were used as primary antibodies. β-actin (1:1000, G8795, Sigma, Saint Louis, MO, USA) was selected as an internal control. The blots were detected with an Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA). The final results were expressed as fold changes compared with the control values.
3
Profiling Histone Modifications and Neuronal Markers
4
Hippocampal Protein Expression Analysis in Injured Mice
5
Immunohistochemical Analysis of Epilepsy Model
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A total of six rats, including three control rats and three rats from pilocarpine model of epilepsy group were used for the immunohistochemistry study. Rats were sacrificed under deep ketamine and xylazine anesthesia 4 h after the first observed seizure (3–5 stage) in chronic phase. 0.3% sodium sulphide in 0.1 M phosphate buffer (PB) was used to perfusion and 4% formaldehyde in phosphate buffer was used for fixation. The fixed brains were cryoprotected with 30% sucrose and cut using a freezing microtome at a thickness of 8 µm.
For immunohistochemistry staining, hippocampus coronal sections were treated with 0.5% Triton X-100 and 3% hydrogen peroxide for 10 min, and then with normal goat serum (1:10). The specimens were incubated with the primary antisera (anti-ADPRC1 from SANTA CRUZ Biotechnology, USA and anti-SNAP 25, anti-calreticulin and anti-PGP 9.5 from Abcam, USA) at room temperature overnight. After proper washing (3*5 min), slides incubated with anti-mouse and rabbit fluorescent secondary antibody for 2 h. After washing step nuclear staining has been done by DAPI. Slides were washed (3*2) and mounted properly for studding by fluorescent microscope.
For immunohistochemistry staining, hippocampus coronal sections were treated with 0.5% Triton X-100 and 3% hydrogen peroxide for 10 min, and then with normal goat serum (1:10). The specimens were incubated with the primary antisera (anti-ADPRC1 from SANTA CRUZ Biotechnology, USA and anti-SNAP 25, anti-calreticulin and anti-PGP 9.5 from Abcam, USA) at room temperature overnight. After proper washing (3*5 min), slides incubated with anti-mouse and rabbit fluorescent secondary antibody for 2 h. After washing step nuclear staining has been done by DAPI. Slides were washed (3*2) and mounted properly for studding by fluorescent microscope.
6
Evaluation of Neuronal and Glial Markers
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The following antibodies were used for this study: anti-NeuN (Millipore, MA); anti-synaptophysin, anti-GFAP, anti-β-Actin, anti-total ERK1/2, anti-phospho ERK1/2 (Cell Signaling, MA); anti-Iba-1 (Wako Chemicals USA, VA); anti-4-HNE (R&D Systems, Minneapolis, MN); anti-Gpx4 (in-house); anti-caspase-3 (Santa Cruz Biotechnology, CA); and anti-SNAP25 (AbCam, Cambridge, MA). Brain regions were dissected then homogenized in 1× RIPA buffer (20 mM Tris, pH7.4, 0.25 M NaCl, 1 mM EDTA, 0.5% NP-40, 50 mM sodium fluoride) with protease and phosphatase inhibitors (Roche, IN). In brief, 30 µg total protein per sample was separated by SDS-PAGE and transferred to PVDF membranes. Membranes were blocked with 5% BSA then incubated with primary antibody overnight at 4 °C. After incubation with fluorophore conjugated secondary antibodies (ThermoFisher, MA) for 1 h, bands were detected using an Odyssey scanner (LI-COR, Lincoln, NE). Signals were analyzed using NIH ImageJ software and normalized to that of β-Actin loading controls.
7
Profiling Histone Modifications and Neuronal Markers
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Antibodies included Anti-H3 (Abcam ab1791), Anti-H3K9ac (Abcam ab4441), Anti-H3K27ac (Abcam ab4729), Anti-H3K122ac (Abcam ab33308), Anti-H4 (Millipore 05-858), Anti-H4K5ac (Millipore 39-584), Anti-H4K12ac (Abcam ab1761), Anti-ACSS2 (T) (Thermo MA5-145810), Anti-ACSS2 (CS) (Cell Signaling 3658), Anti-ACL (Proteintech 15421-1-AP), Anti-α-Tubulin (Sigma T8328), Anti-GAPDH (Fitzgerald Industries 10R-G109A), Anti-KAT3A/CBP (Abcam ab2832), Anti-SNAP25 (Abcam ab5666), Anti-Synaptophysin (Millipore MAB368), Anti-MAP2 C/D (Cell Signaling #8707), Anti-NR4A2 (Santa Cruz sc-991), Anti-NeuN (Millipore ABN78)
8
Antibodies for Synaptic Protein Analysis
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The following antibodies were used for western blot: Anti-neuroserpin goat polyclonal antibody G64 (generation and affinity-purification have been previously described13 (link)) (0.5 ug/ml); Anti-synaptophysin (Abcam ab32594, 1:1000); Anti-SNAP25 (Abcam ab41455, 1:1000); Anti-neuroserpin (Abcam ab46761, 1:5000 and ab32901, 1:2000); Anti-synapsin-I (Cell Signaling Technology D12G5, 1:1000); Anti-PSD-95 (Millipore clone EP2652Y, 1:1000); Anti-beta-actin (Millipore clone C4, 1:5000); Anti-PDI (StressMarq SPC114C, 1:2000); Anti-GFP (Clontech mouse Living Colors monoclonal antibody 632,459, 1:5000). The following antibodies were used for immunofluorescence: Anti-MAP2 (Sigma M4403, 1:200); Anti-cleaved caspase 3 (R&D Systems AF835, 1:100); Anti-synaptophysin (Abcam ab32594, 1:200); Anti-PSD-95 (Millipore MAB1598, 1:100); Anti-neuroserpin goat polyclonal antibody G64 (3 ug/ml).
9
Reagents for Synaptic Protein Analysis
10
Hippocampal Protein Expression Analysis in Injured Mice
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Ipsilateral hippocampal tissues from sham and CCI injured mice were homogenized in RIPA buffer with protease and phosphatase inhibitor cocktail (Sigma-Aldrich, St. Louis, MO) and benzonase nuclease (Millipore Corporation, Billerica, MA) and mixed by rocking at 4 °C for at least 15 min. Tissues were centrifuged, supernatant was recovered, samples were diluted and standardized to protein concentrations. Protein samples were separated on 8–10% SDS-PAGE gel and transferred to a nitrocellulose membrane. Membranes were then blocked with 5% milk or 5% BSA in 0.1 M phosphate buffer with 0.1% Tween-20 for 1 h at room temperature (RT) and incubated overnight at 4 °C with primary antibodies. Membranes were incubated for 1 h at RT with HRP-conjugated secondary antibodies (Jackson Immunoresearch Laboratories, West Grove, PA). Bands were visualized using SuperSignal substrate (ThermoScientific, Pittsburg, PA). The following primary antibodies were used: anti-GluR1, anti-NR1, anti-NR2B (EMD Millipore, Billerica, MA), anti-GFAP (BD Biosciences, San Jose, CA), anti-SNAP25, anti-SNAP23 (ABCAM, Cambridge, MA) and anti-β-tubulin (Sigma-Aldrich, St. Louis, MO) antibodies. ImageJ was used to perform density analysis. Protein measurements were standardized to β-tubulin and normalized to average WT sham signals.
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