The hippocampi of rats were rapidly collected and stored in liquid nitrogen immediately. The protein concentration was tested using a Pierce bicinchoninic acid (BCA) protein assay kit (Thermo scientific). Samples containing 40 μg of total protein were separated in 10%–15% sodium dodecyl sulfate (SDS)‐polyacrylamide gels and then transferred to polyvinylidene difluoride (PVDF) membranes (0.22 μm; Millipore Corp). The membranes were blocked in TBST containing 5% nonfat dry milk for 90 min and incubated with the following primary antibodies overnight at 4°C: anti‐PSD95 (goat monoclonal antibody; Abcam, 1:2,000), anti‐SYP (rabbit monoclonal antibody; Abcam, 1:2,000), anti‐GAP43 (rabbit monoclonal antibody; Abcam, 1:2,000), anti‐BDNF (rabbit monoclonal antibody; Abcam, 1:2,000), anti‐p‐CREB (phosphor S133, rabbit monoclonal antibody; Abcam, 1:5,000), anti‐CREB (rabbit monoclonal antibody; Abcam, 1:2,000), and anti‐β‐actin antibody (1:500, mouse monoclonal antibody; Boster, 1:500). On the next day, the membranes were incubated with the corresponding secondary antibody (anti‐goat/rabbit/mouse IgG; ZSGB‐BIO, 1:5,000) for 90 min at room temperature. After using clarity western electrochemiluminescence (ECL) substrate (Bio‐Rad), protein bands were visualized in the Bio‐Rad Imager. The intensities of the protein bands were analyzed by densitometry using Syngene imaging systems.
Anti syp
Manufactured by Abcam
Sourced in United States, China
Anti-SYP is a laboratory reagent used for the detection and identification of the synaptic protein Synaptophysin (SYP) in biological samples. Synaptophysin is a membrane protein found in presynaptic vesicles and is commonly used as a marker for synapses.
Automatically generated - may contain errors
Lab products found in correlation
Anti psd95, by Abcam (3 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ecl kit, by Thermo Fisher Scientific (2 mentions)
Anti gap43, by Abcam (1 mentions)
Anti pcreb, by Abcam (1 mentions)
Anti β actin antibody, by Boster Bio (1 mentions)
Clarity western electrochemiluminescence ecl substrate, by Bio-Rad (1 mentions)
Imager, by Bio-Rad (1 mentions)
Anti creb, by Abcam (1 mentions)
Anti bdnf, by Abcam (1 mentions)
Np0322box, by Thermo Fisher Scientific (1 mentions)
Anti ldh, by Santa Cruz Biotechnology (1 mentions)
Anti β actin antibody, by Santa Cruz Biotechnology (1 mentions)
Anti ldha, by Santa Cruz Biotechnology (1 mentions)
Scion image software, by Techcomp Instruments (1 mentions)
Anti gfap, by Santa Cruz Biotechnology (1 mentions)
Anti akt, by Cell Signaling Technology (1 mentions)
Anti β actin, by Boster Bio (1 mentions)
Anti p akt, by Cell Signaling Technology (1 mentions)
8 protocols using anti syp
1
Hippocampal Protein Expression Analysis
2
Quantifying Cell-Specific Protein Expression
3
Western Blot Analysis of Hippocampal Proteins
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Hippocampus tissues were homogenized on ice for 30 min and centrifuged at 12000 rpm at 4 °C for 15 min. The supernatants were collected for follow-up measurement. Protein concentration was determined using BCA method. Equal amounts of protein (25 μg) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. After blocked in 5% nonfat milk/tris-buffered saline with Tween 20 (TBST) or 5% bovine serum albumin (BSA) at RT for 1 h, the membranes were incubated overnight with primary antibodies including anti-GLT-1 (1:1000; Millipore), anti-PSD-95 (1:1000; Abcam), anti-SYP (1:1000; Abcam), anti-NR1 (1:1000; Cell Signaling Technology), anti-NR2A (1:1000; Cell Signaling Technology), anti-NR2B (1:1000; Cell Signaling Technology), anti-PI3K (1:1000; Abcam), anti-Akt (1:1000; Cell Signaling Technology), anti-p-Akt (1:1000; Cell Signaling Technology), anti-Bax (1:1000; Abcam), anti-Bcl-2 (1:2000; Abcam) and anti-β-actin (1:500; Boster ). Then the membranes were washed with TBST for 3 × 10 min and incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1:5000; Servicebio) at RT for 1 h. Next, the membranes were washed for 3 × 10 min and the immunoreactive bands were detected by the Bio-Rad Chemi Doc XRS + imaging system. Densitometric analysis was analyzed with the use of ImageJ software.
4
Protein Extraction and Western Blot Analysis
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The extraction of total proteins from brain tissues was completed with RIPA buffer (Cell Signaling Technology, Boston, MA, USA). A BCA Protein Quantification Kit (Key GEN Biotech, Nanjing, China) was used to measure the protein concentration. Protein samples of the same amount (30 μg) were separated by SDS-PAGE and transferred to PVDF membranes (Millipore, Billerica, MA, USA). The membranes were sealed with 5% milk and combined with anti-NLRP3 (1:1000, #15101, Cell Signaling Technology, Boston, MA, USA), anti-SYP (1:1000, #ab52636, Abcam, Cambridge, UK), anti-PSD95 (1:5000, #ab2723, Abcam, Cambridge, UK), anti-β-actin (1:1000, #4970, Cell Signaling Technology, Boston, MA, USA), anti-GFAP (1:1000, #ab68428, Abcam, Cambridge, UK), anti-Iba-1 (1:1000, #ab178846, Abcam, Cambridge, UK) at 4 °C overnight. Next, we used the corresponding secondary antibody to incubate the film at room temperature for 2 h. Immune complexes were assessed with an ECL kit (Thermo Scientific, Waltham, MA, USA), and the results were quantified by ImageJ.
5
Immunohistochemical Analysis of SYP and TET3
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Paraffin-embedded 4-mm-thick specimens were dewaxed in turpentine and rehydrate through decreased concentrations of ethanol. Antigen retrieval was not performed. Endogenous peroxidase activity was blocked by using 3% H2O2 in methnal for 10min and then soaked with phosphate buffered saline (PBS) (pH 7.2–7.4) three times for 5 min. The sections were then pre-incubated with 0.25% trypsin-EDTA for 10 min to block non-specific antigens. The tissue sections were allowed to react overnight at 4°C with anti-SYP (dilution 1/100; Abcam) TET3 (dilution 1/200, Abcam) antibodies. The slides were then incubated at room temperature for 1h, rinsed with PBS three times and incubated with appropriate biotinylated secondary antibodies for 30 min. For the quantification of immunohistochemistry, 10 fields of each section were viewed and analysed with an image analysis program (Bioquant, Nashville, TN).
6
Immunoblotting of Cellular Proteins
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Protein samples were lysed in 5 × SDS sample buffer containing beta-mercaptoethanol, boiled at 95°C for 10 min and loaded into each well for SDS-PAGE. Samples were then transferred to PVDF membrane (Thermo) and immunoblotted using anti-NDUFA9 (Invitrogen), anti-NDUFS3 (Invitrogen), anti-NDUFA13 (Invitrogen), anti-ATP5A (Invitrogen), anti-SDHA (CST), anti-VDAC (CST), anti-HSP60 (CST), anti-PHB1 (CST), anti-PDH (CST), anti-GAPDH (Sigma), anti-GRP78 (SCBT), anti-ABCA1 (SCBT), anti-FLOT1 (CST), anti-STAT3 (CST), anti-STAT1 (SCBT), anti-AMPK (CST), anti-ERK1/2 (CST), anti-p38 (CST), anti-SYP (Abcam), anti-ACSL4 (Abcam), all diluted in 5% BSA:TBST at 1:1000, followed by appropriate HRP-conjugated secondary antibodies (Thermo) incubation (diluted in 5% non-fat milk:TBST at 1:10,000), and developed using the SuperSignalTM West Femto Maximum Sensitivity Substrate (Thermo).
7
Immunohistochemical Analysis of Spinal Cord Tissues
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Spinal cord tissues were fixed in 10% formalin, embedded in paraffin, and then sectioned. Tissue sections were dewaxed in xylene and rehydrated in a series of ethanol concentrations. Immunohistochemical staining was performed according to the manufacturer’s protocols (Boster, Wuhan, China). To the stain spinal cord tissues, sections were incubated with various primary antibodies at 4°C overnight, including anti-Gap-43(1:100, CST, United States), anti-NF200 (Boster, China), anti-SYP (1:100, Abcam, United States), and anti-PSD95 (Boster, China). The following morning, the sections were rinsed in PBS and then incubated with biotinylated polyclonal secondary antibodies. Finally, the sections were washed and incubated with horseradish peroxidase-labeled streptavidin. Sections were then developed with diaminobenzidine and counterstained with hematoxylin. Representative images were acquired using a digital microscope (Nikon, Tokyo, Japan).
8
Analysis of Hippocampal Synaptic Proteins
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Proteins were isolated from the hippocampal neurons and were resolved with 10% SDS-PAGE gels on the eleventh day in vitro. After migration, they were transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA) and blocked in tris-buffered saline containing 0.2% tween-20 and 5% skim milk powder for 1 h at room temperature. Then, the membrane was incubated overnight at 4 °C with the primary antibodies at specific dilutions as follows: anti-GluN1 (1:1,000, Abcam), anti-GluA1 (1 μg/mL, Abcam), anti-Syp (1:20,000, Abcam), anti-PSD-95-specific (1:1,000, Proteintech), anti-active-β-catenin (anti-ABC; 1:1,000, Millipore), and anti-Wnt7a (1:500, Proteintech). β-Tubulin (1:1,000, Cell Signaling Technology, Danvers, MA) was used as the gel loading control. After incubation with primary antibodies overnight at 4 °C, the blots were then incubated with a horseradish peroxidase-conjugated secondary antibody (1:3,000, Proteintech) for 1 h at room temperature. An enhanced chemiluminescence reagent was used to detect the proteins. Quantity One software was used for the analysis of band intensity.
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