The protocol for immunohistochemistry as previously described.47 The retinas were incubated in PBS with 1% Triton X-100 and 0.5% Tween 20 for 1 h at room temperature and in 4% BSA for 1 h at room temperature and then incubated overnight at 4°C with primary antibodies: RBPMS (1:500, Abcam), ChAT (1:100, Abcam), VGlut2 (1:100, Frontier Institute), syntaxin (1:100, Abcam), tyrosine hydroxylase (1:100, Abcam) and Prox1 (1:100, AngioBio) in blocking buffer. Secondary anti-rabbit, mouse, and goat IgG, conjugated with Alexa TM488, TM594, and 633, respectively (1:1,000; Molecular Probes), were applied for 1 h at room temperature. The retinas then were flat mounted, and the sections were mounted on slide glass. The TUNEL assay was performed based on our previous reports.48 (link),49
Syntaxin
Manufactured by Abcam
Syntaxin is a membrane-associated protein that plays a crucial role in the regulation of vesicle-mediated exocytosis. It is a component of the SNARE (Soluble N-ethylmaleimide-sensitive factor Attachment Protein Receptor) complex, which is responsible for the fusion of vesicles with the target membrane. Syntaxin functions as a receptor for the SNARE machinery, enabling the docking and subsequent fusion of vesicles with the plasma membrane.
Automatically generated - may contain errors
Lab products found in correlation
Tyrosine hydroxylase, by Abcam (1 mentions)
Alexa 488, by Thermo Fisher Scientific (1 mentions)
Rbpms, by Abcam (1 mentions)
Sirt1, by Abcam (1 mentions)
Map2b, by BD (1 mentions)
Alexa fluor 488 green alexa fluor 594 red conjugated secondary antibody, by Abcam (1 mentions)
Fibroblast growth factor 2 (fgf2), by Abcam (1 mentions)
Erbb4, by Abcam (1 mentions)
Synaptophysin, by Merck Group (1 mentions)
Biii tubulin, by Merck Group (1 mentions)
Mem media, by Corning (1 mentions)
Non fat dry milk, by GE Healthcare (1 mentions)
Bradford assay, by Merck Group (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Protease inhibitor, by Roche (1 mentions)
3 protocols using syntaxin
1
Immunohistochemistry Protocol for Retina Analysis
2
Immunostaining of Brain Sections and Neurons
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Brain sections or glass coverslips were xed with 4% paraformaldehyde for 30 min, rupture of cell membrane was performed using 0.1% Triton X-100 for 10 min at room temperature, PBS containing 5% normal goat serum for 1 h at room temperature was used for blocking, and sections or coverslips were incubated overnight at 4 °C with the following primary antibodies: ErbB4, NRG, FGF2, Sirt1, syntaxin, Homer, and MAP2 (Abcam). Alexa Fluor 488 (green)/Alexa Fluor 594 (red) conjugated secondary antibody (Abcam, Cambridge, MA) were then used to detect primary antibodies for 1 h.
For dendritic spine analysis, the primary neurons coverslips were incubated with the primary antibodies: microtubule-associated protein 2B (MAP2B; 1:200; BD Transduction Laboratories, San Jose, CA, USA) and vesicular glutamate transporter 1 (vGlut1; 1:100; Neuromab, Davis, CA, USA) overnight at 4 °C. Alexa Fluor 488 (green)/Alexa Fluor 594 (red) conjugated secondary antibody (Abcam, Cambridge, MA) were then used to detect primary antibodies for 1 h. At least 10 cultured primary neurons per coverslip were used for quantitative analysis.
For dendritic spine analysis, the primary neurons coverslips were incubated with the primary antibodies: microtubule-associated protein 2B (MAP2B; 1:200; BD Transduction Laboratories, San Jose, CA, USA) and vesicular glutamate transporter 1 (vGlut1; 1:100; Neuromab, Davis, CA, USA) overnight at 4 °C. Alexa Fluor 488 (green)/Alexa Fluor 594 (red) conjugated secondary antibody (Abcam, Cambridge, MA) were then used to detect primary antibodies for 1 h. At least 10 cultured primary neurons per coverslip were used for quantitative analysis.
3
Hippocampal Slices Immunoblotting Analysis
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The hippocampal slices were obtained from 8-to 12-week-old C57BL/6 male mice killed by decapitation. The temporal lobes were cut into 350-mm-thick slices with vibratome. The hippocampal slices were then cultured for immunoblotting in MEM media (Corning) and treated with 5-mM GlcNAc and 150-mM UDP-GlcNAc or 50-mg/mL Ab 25e35 for 1 week. Slices were then homogenized in RIPA buffer containing protease inhibitors (Roche Diagnostics). After 2 hours at 4 C, homogenates were centrifuged at 4000 rpm to discard cellular debris. Protein content was determined by Bradford Assay (Sigma-Aldrich). Protein lysates were diluted in Laemli buffer, boiled at 90 C for 5 minutes, and then resolved by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins were transferred onto the nitrocellulose membrane (Bio-Rad), which were blocked with Tris-buffered saline and 0.1% Tween 20% and 10% nonfat dry milk (GE-Healthcare). Membranes were then incubated at 4 C overnight with the following primary antibodies: syntaxin (Abcam), synaptophysin (Millipore), bIII tubulin, and actin (Sigma-Aldrich). Appropriate secondary IgG HRP-conjugated (GE Healthcare) was added for 1 hour, and chemiluminescent detection was performed with ECL Plus advanced (Amersham and GE Healthcare). Quantitative analysis of the signal obtained was performed by Image J software (National Institutes of Health).
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