Nerve segments were homogenized and extracted using RIPA lysis buffer containing protease/phosphatase inhibitor (GenDEPOT) and lysates were sonicated ten times for 30 s. The homogenized sample was centrifuged at 15,000 rpm for 10 min at 4 °C and the cleared lysates were quantified using a BCA protein assay kit (GenDEPOT). Equal amounts of protein were separated on 12% SDS-PAGE gels and transferred to a polyvinylidene fluoride (PVDF) membrane (Bio-Rad). Blots were probed with antibodies against P0 (1:1000, ab31851; Abcam) and MAG (1:1000, 34-6200; Invitrogen). β-actin (1:1000, 4967; Cell Signaling Technology) was used as a loading control. Protein expression was quantified using ImageJ software.
Ab31851
Manufactured by Abcam
Sourced in United States
Ab31851 is a recombinant antibody that recognizes the protein SNAP25. It is produced in a mammalian expression system and purified by Protein A affinity chromatography. The antibody is suitable for use in immunohistochemistry and western blotting applications.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Cell Signaling Technology (2 mentions)
Ab228549, by Abcam (2 mentions)
Ab4680, by Abcam (2 mentions)
Ab150088, by Abcam (2 mentions)
Ab197485, by Abcam (2 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Ab52987, by Abcam (1 mentions)
Ab16667, by Abcam (1 mentions)
4 6 diamino 2 phenyl indole dapi, by Vector Laboratories (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Hrp linked anti rabbit igg, by Cell Signaling Technology (1 mentions)
Hrp linked anti mouse igg, by Cell Signaling Technology (1 mentions)
Vin 11 5, by Merck Group (1 mentions)
Ab62631, by Abcam (1 mentions)
Ab89780, by Abcam (1 mentions)
Cy3 conjugated goat anti rabbit secondary antibody, by Merck Group (1 mentions)
Axio imager m2, by Zeiss (1 mentions)
Phospho jnk, by Cell Signaling Technology (1 mentions)
Phospho p38, by Cell Signaling Technology (1 mentions)
M3821, by Merck Group (1 mentions)
11 protocols using ab31851
1
Peripheral Myelin Protein Analysis
2
Immunofluorescence Analysis of Peripheral Nerve
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Teased nerve fibers mounted on slides were treated with PBS containing 4% Paraformaldehyde for 30 min and blocked with PBS containing 0.2% Triton X-100 and 2% BSA for 60 min. Nerve fibers were incubated with primary antibody (anti-p75NTR antibody 1:1000, ab52987, Abcam; anti-myelin protein zero (MPZ) antibody, 1:1000, ab31851, Abcam; anti-c-JUN antibody, 1:1000, #9165, Cell Signaling; anti-Ki67 antibody,1:100, ab16667, Abcam) for 16 h at 4°C and washed three times with PBS. Next, slides were incubated with Alexa 549- or 488-conjugated secondary antibody (1:800, Alexa Fluor) for 2 h at room temperature and washed three times with PBS. Finally, slides were incubated with PBS counterstained with 4′6-diamino-2-phenyl indole (DAPI; Vectashield, Vector Laboratories) to visualize nuclei. DAPI staining was used for enumeration and identification of nuclei. The slides were visualized using a 20×/0.50 Plan-Neofluar lens (Carl Zeiss). c-JUN-positive endonuclear cells were counted to analyze percent of c-JUN (+)in three independent experiments. Ki67-positive endonuclear cells were counted to analyze percent of Ki67 (+)in three independent experiments.
3
Western Blotting Analysis of Myelination Markers
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Total protein was extracted from purified SCs or sciatic nerves that were washed with PBS 3 times and dissociated in lysis buffer containing 10 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA-2Na, 1% Nonidet P-40, 1 mM Na2VO4, 10 mM NaF and Protease Inhibitor Cocktail (11873580001, Roche) for 30 min on ice. The supernatants were collected by centrifugation. Total protein was denatured and used for western blotting. The following primary antibodies were used in western blots: anti-rabbit Myelin Protein Zero (ab31851, Abcam), anti-rat myelin basic protein (MAB386, Merck Millipore), anti-mouse MAG (sc-166848, Santa Cruz Biotechnology), anti-rabbit TRPV4 (CB-ACC-034, Alomone), anti-rabbit EGR2 (ab108399, Abcam), anti-rabbit c-Jun mAb (9165, Cell Signaling Technology), and anti-mouse vinculin antibody (VIN-11-5, Sigma). The following secondary antibodies were used for western blotting: HRP-conjugated GAPDH rabbit mAb (8884, Cell Signaling Technology), HRP-linked anti-rabbit IgG (7074, Cell Signaling Technology), HRP-linked anti-mouse IgG (7076, Cell Signaling Technology), and HRP-linked anti-rat IgG (112-035-062, Jackson).
4
Immunostaining and Imaging of Myelination in Co-cultured DRGs and Schwann Cells
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Co-cultured DRGs and Schwann cells and crushed proximal nerve stumps were collected and processed as previously described (Zanazzi et al., 2001). For immunostaining, cells and tissue slices were fixed with 4% paraformaldehyde/PBS for 5 minutes at room temperature, blocked with 5% goat serum for 30 minutes at room temperature and incubated with primary antibodies: anti-myelin basic protein (MBP) (1:500; AB62631, Abcam St Louis, MO, USA), anti-peripheral myelin protein zero (P0) (1:125; AB31851, Abcam), or anti-myelin-associated glycoprotein (MAG) (1:200; AB89780, Abcam) overnight at 4°C. Specimens were then incubated with Cy3-conjugated goat anti-rabbit secondary antibody (1:1,000; Sigma) for 2 hours at room temperature, and were then mounted on microscope slides. Samples were visualized and images captured using an optical and epifluorescence microscope (Axio Imager M2, Carl Zeiss Microscopy GmbH, Jena, Germany). All assays were performed in triplicate.
5
Immunoblotting Analysis of Cellular Signaling
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Cells were homogenized in 100 μL Kaplan buffer (150 mM NaCl, 50 mM Tris-HCl (pH 7.4), 1% NP-40, 10% glycerol, and a protease inhibitor cocktail (Roche Diagnostics)). The lysate was subject to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and WB analysis with a standard procedure [62 (link)] using antibodies against AKT (1:1000; CST 4691; Cell Signaling Technology, Beverly, MA, USA), phospho-AKT (1:1000; CST 4056; Cell Signaling Technology), ERK1/2 (1:1000; CST 4695; Cell Signaling Technology), phospho-ERK1/2 (1:1000; CST 9101; Cell Signaling Technology), p38 (1:1000; CST 9212; Cell Signaling Technology), phospho-p38 (1:1000; CST 9215; Cell Signaling Technology), JNK (1:1000; CST 9252; Cell Signaling Technology), phospho-JNK (1:1000; CST 4668; Cell Signaling Technology), c-JUN (1:1000; CST 9165; Cell Signaling Technology), phospho-c-JUN (1:1000; CST 3270; Cell Signaling Technology), MBP (1:1000; M3821; Sigma-Aldrich), MAG (1:1000; MAB1567; Chemicon, Temecula, CA, USA), P0 (1:1000; ab31851; Abcam, Cambridge, UK), HIF1α (1:1000; CST 14179; Cell Signaling Technology), and β-actin (1:1000; CST 4970; Cell Signaling Technology). Protein expression levels were determined using the MF-ChemiBIS 3.2 imaging system (Berthold Technologies, Bad Wildbad, Germany).
6
Comprehensive Protein Expression Analysis
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Western blotting was performed in accordance with a previously published method (Chen et al., 2019 (link)). Total proteins were extracted by RIPA lysis buffer with proteinase inhibitor (Roche, Switzerland). In total, 30 μg aliquots of protein were separated in 10% SDS-PAGE gels and then transferred onto PVDF membranes (Millipore, United States). After 1.5 h of blocking with 5% w/v nonfat dry milk buffer, the membrane was incubated overnight with primary antibodies against GM130 (#66662-1-Ig, Proteintech), GAPDH (#10494-1-AP, Proteintech), CD63 (#PA5-92370; Invitrogen), CD9 (#ab92726, Abcam), CD81 (#ab109201, Abcam), S100ß (#ab52642; Abcam), nerve growth factor receptor (NGFR, #8238, Cell Signaling Technology), myelin protein zero (MPZ, #ab31851, Abcam), glial fibrillary acidic protein (GFAP, #3670, Cell Signaling Technology), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit delta (PIK3CD, #ab109006, Abcam) and phosphorylated serine/threonine kinase (p-AKT, #4060, Cell Signaling Technology). Then, the membrane was incubated with secondary antibodies (Aspen, China) and exposed to X-ray film (UVP, United States).
7
Myelin Staining and Immunohistochemistry Protocol
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The slices were left to dry at RT for 1 h and washed once with 0.01 M of PBS. In order to stain myelin effectively, slices were placed in ascending (50, 70, 90, 95, and 100%) and then descending ethanol dilutions (100, 95, 90, 70, and 50%). Again, the slices were washed with 0.01 M of PBS and subsequently blocked in 10% normal donkey serum (NDS) dissolved in 0.01 M of PBS with 0.1% Triton X-100 for 30 min. Primary antibodies diluted by 0.01 M of PBS with 0.1% Triton X-100 were applied to the sections overnight. The next morning, slices were washed with PBS and then incubated by secondary antibodies (Alexa Fluorescence 488, 594, and 647, Abcam, United Kingdom) diluted by 0.01 M of PBS with 0.1% Triton X-100 for 2 h. The slices were washed again and cover-slipped using Fluoroshield (ab104135, Abcam, United Kingdom). The antibodies used were directed against the following antigens: glial fibrillary acidic protein (GFAP) (1:1,000, Cell Signaling Technology, 3,670), myelin basic protein (MBP) (1:500, Abcam, ab4039), SMI312 (1:1,000, Covance, SMI-312R-100), NeuN (1:800, Abcam, ab177487), P0 (1:200, Abcam, ab31851), CC1 (1:300, Abcam, ab16794), and Olig2 (1:500, Millipore, AB9610).
8
Sciatic Nerve Histological Analysis
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The full length of the sciatic nerve was harvested after compression tube removal and fixed in 4% paraformaldehyde (PFA) at 4 °C overnight. The nerves were embedded in paraffin, and cross-sections were taken in the middle portions of the compression area. The 5 µm cross sections were taken from the embedded blocks and mounted on slides. The nerve sections were deparaffinized and serially rehydrated using xylene and ethanol. Antigen retrieval was performed using 0.01 M citrate buffer (pH 6.0), which was microwaved for 15 min. Permeabilization and blocking of nonspecific binding were performed with Tween 20 and 1:20 diluted goat serum (ab7481, Abcam, Cambridge, UK), respectively. Primary antibody staining was performed with anti- NF heavy chain antibody (1:500, ab4680; Abcam) and anti-P0 antibody (1:200, ab31851; Abcam) in 5% BSA. Incubation with 4′,6-diamidino-2-phenylindole (DAPI, 1:1000, ab228549; Abcam) and fluorescent secondary antibodies (Alexa Fluor 488, and 594-conjugated antibodies [1:500, ab197485 and ab150088, Abcam]) was performed after washing the sections in PBS to remove the primary antibodies. Fluorescent images were captured with a Leica DMI4000B fluorescence microscope (Leica Microsystems, Wetzlar, Germany) and semiautomatic analysis was performed with ImageJ (U.S. National Institutes of Health) to determine the number of NF- or P0 expressing axons.
9
Sciatic Nerve Immunohistochemistry in Rats
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On the 14th postoperative day, rats were anesthetized with sodium pentobarbital and perfused transcardially with 0.9% saline. The left sciatic nerve segment was excised carefully and fixed in 4% paraformaldehyde for 6 h, subsequently dehydrated in 20% and 30% sucrose at 4°C overnight, and embedded in OCT. After slicing, sections were air-dried overnight and stored at -40°C. The frozen sections were equilibrated to room temperature for 10 min and washed in 0.01 M PBS for 10 min, then treated with citrate buffer at 95°C for 5 min and cooled to room temperature. The tissue sections were blocked with immunofluorescence blocking solution containing 0.3% Triton X-100 for 1 hour at room temperature. Afterwards, the sections were incubated with primary antibodies against S100 (ab868, 1 : 200; Abcam) and P0 (ab31851, 1 : 100; Abcam) overnight at 4°C, washed, and then incubated with fluophore-conjugated secondary antibodies (Cell Signaling Technology) for 1 hour at room temperature. Tissues were covered with mounting medium including 4′,6-diamidin-2-phenylindol solution (DAPI) and imaged by a fluorescence microscope (Nikon DS-Ri2).
10
Sciatic Nerve Regeneration Analysis
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Twelve weeks postoperatively, mice were euthanized by CO2 asphyxiation with cervical dislocation. The full length of the sciatic nerve containing the autograft was harvested and xed in 4% paraformaldehyde (PFA) at 4°C overnight. The nerves were embedded in para n, and cross-sections were taken from the proximal and central portions of the autograft. Cross-sections (5 µm) were taken from the embedded blocks and mounted on slides. The sections were depara nized and serially rehydrated using xylene and ethanol, respectively. Antigen retrieval, permeabilization and blocking of nonspeci c binding was performed. Primary antibody staining was performed with anti-NF heavy chain antibody (1:500, ab4680; Abcam) and anti-P0 antibody (1:200, ab31851; Abcam) in 5% BSA. Incubation with 4,6-diamidino-2phenylindole (DAPI, 1:1000, ab228549; Abcam) and uorescent secondary antibodies (Alexa Fluor 488 and 594-conjugated antibodies [1:500, ab197485 and ab150088, Abcam]) was performed after washing the sections in PBS to remove the primary antibodies. The nerve sections were examined using a Leica DMI4000B uorescence microscope (Leica Microsystems, Wetzlar, Germany). Four stained nerve sections per group were analyzed in a semiautomatic fashion using ImageJ (National Institutes of Health, Bethesda, MD) to determine the number of NF-or P0-expressing axons.
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