Immunohistochemistry of skeletal muscle was performed as previously described (Liu et al., 2017 (link)). For immunohistochemistry of fixed frozen sections, the following antibodies were used: Twist2 (Abcam, ab66031, 1:200), Sema3a (Abcam, ab23393, 1:100), Nrp1 (Abcam, ab81321, 1:100), and Myh4 (Invitrogen, 14-6503-82, 1:100). For immunohistochemistry of raw embedded frozen sections, the following antibodies were used: Twist2 (Abcam, ab66031, 1:200), Sema3a (Abcam, ab23393, 1:100), Nrp1 (Abcam, ab81321, 1:100), myosin IIa (Developmental Studies Hybridoma Bank, SC-71, 1:10), myosin IIb (Developmental Studies Hybridoma Bank, BF-F3, 1:10), Slow myosin (Sigma-Aldrich, NOQ7.5D, 1:250), and Fast myosin (Sigma-Aldrich, clone My32, 1:250). Alexa Fluor secondary antibodies were used according to the manufacturer’s instructions. TdTO signals were detected by direct fluorescent imaging. Wheat germ agglutinin staining was performed on both frozen and paraffin-embedded sections, using WGA-Alexa Fluor 555 (W32464) or WGA-Alexa 647 conjugate (W32466) (Life Technologies, 50 mg ml−1). Images were taken on a Zeiss LSM700 confocal microscope. Muscle fiber numbers were quantified by ImageJ.
Sema3a
Manufactured by Abcam
Sourced in United States, United Kingdom
Sema3A is a recombinant protein that represents the semaphorin 3A domain. Semaphorins are a family of secreted and membrane-bound proteins involved in axon guidance and neuronal development. Sema3A functions as a chemorepellent, guiding the navigation of neuronal growth cones during neural development.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (2 mentions)
Bf f3, by Merck Group (1 mentions)
Myosin iib, by Developmental Studies Hybridoma Bank (1 mentions)
Slow myosin, by Merck Group (1 mentions)
Ab23393, by Abcam (1 mentions)
Lsm 700 confocal microscope, by Zeiss (1 mentions)
Ab66031, by Abcam (1 mentions)
Fast myosin, by Merck Group (1 mentions)
Sc 71, by Developmental Studies Hybridoma Bank (1 mentions)
Gapdh, by Abcam (1 mentions)
Beyoecl plus kit, by Beyotime (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Micro bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Gapdh, by Merck Group (1 mentions)
Bcl 2, by Cell Signaling Technology (1 mentions)
Proteinase inhibitor cocktail, by Roche (1 mentions)
Mmp13, by Abcam (1 mentions)
Bradford protein assay, by Bio-Rad (1 mentions)
Iblot system, by Thermo Fisher Scientific (1 mentions)
Il 1β, by Cell Signaling Technology (1 mentions)
9 protocols using sema3a
1
Immunohistochemical Analysis of Skeletal Muscle
2
Western Blot Analysis of Sema3A and Nrp-1
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Samples were resolved on SDS-PAGE gel. Proteins were transferred onto a PVDF membrane (Millipore, Germany) by conventional methods. It was probed with primary antibodies against sema3a (1:1,000, Abcam), Nrp-1 (1:1,000, CST), GAPDH (1:10,000, Abcam), and then the immunoreactive protein was visualized by using BeyoECL Plus kit (Beyotime, China).
3
Protein Extraction and Western Blot Analysis
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Tissues were lysed in RIPA SDS (50 mM Tris–HCl, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% NP40, Proteinase Inhibitor Cocktail, Roche, 11697498001 and 0.5 mM orthovandate) after which the protein concentration was determined using a Micro BCA Protein assay kit (Thermo Scientific, lot# MJ162220). Samples were diluted to a final concentration of 2 μg/μl after which two times sample buffer (25 ml 0.5 M Tris–HCl, 20 ml 100% glycerol, 20 ml, 20% SDS, 35 ml Aqua Dest with 1:10 β-mercaptoethanol) was added to protein samples 1:1. Cells were directly lysed in sample buffer. For western blot analyses 20 μg protein was loaded on a 10% gel (4 ml Aqua Dest, 3.3 ml 30% AcrylAmide, 2.5 ml 1.5 M Tris–HCl pH 8.8, 0.1 ml 10% SDS, 0.004 ml TEMED). SDS PAGE was performed at 150 V for approximately 90 min after which the gel was transferred to a PVDF membrane by blotting at 200 mA for 2 h. Membranes were blocked with 3.5% Protifar for 1 h. Overnight primary antibody incubation was performed in 5% BSA with antibodies against Sema3A (Abcam, ab23393), Cleaved Caspase 3 (Cell Signaling Technology (CST) #9664S), BCL2 (Cell Signaling Technology (CST) #2870S), GAPDH (Millipore, MAB374).
4
Western Blot Analysis of Protein Expression
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Whole cell lysates were collected by adding Triton X-100 buffer (Roche Diagnostics). Protein concentration was determined by the Bradford protein assay (Bio-Rad Laboratories, Hercules, CA, USA). Equal amounts (30 μg) of protein were run in each lane on a Tris-glycine gel using SDS-PAGE (Atto, Tokyo, Japan). After electrophoresis, the proteins were transferred to a polyvinylidene difluoride (PVDF) membrane using an i-Blot system (Invitrogen). The membrane was then blocked with 4% (w/v) Block Ace (Yukijirushi Nyugyo Inc., Tokyo, Japan) and probed with 1 : 1000 dilutions of Sema3A (Abcam, Cambridge, UK), NRP-1 (Abcam), PLXA1 (ECM Biosciences, Versailles, KY, USA), IL-1β (Cell Signaling Technology Inc., Beverly, MA, USA), MMP-13 (Abcam), phospho-AKT (CST), total-AKT (CST), phospho-ERK1/2 (CST), total-ERK1/2 (CST), phospho-NF-κB (CST), and total-NF-κB (CST) antibodies overnight at 4°C. Next, 1 : 20000 diluted horseradish peroxidase conjugated antimouse antibody or antirabbit antibody (GE Healthcare Life Sciences, Tokyo, Japan) was used as the secondary antibody. Protein expression was normalized to beta-actin. Signals were detected using an Odyssey® Blot Analyzer (M&S Techno Systems, Osaka, Japan).
5
Immunofluorescence Staining of Pancreatic Tumors
6
Hippocampal Protein Analysis in Mice
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Mice anesthetized with 2% pentobarbital sodium solution were sacrificed and the hippocampus was removed and homogenized in radioimmunoprecipitation assay buffer containing protease and phosphatase inhibitor. The homogenate was centrifuged at 13,200 rpm for 30 min at 4°C. The supernatant was collected and protein concentration was measured with a bicinchoninic acid protein assay kit (Thermo Fisher Scientific, Waltham, MA, United States). Protein samples were electrophoretically separated on 4–12% polyacrylamide gels and transferred to a polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA, United States) that was blocked in 5% nonfat milk for 2 h at room temperature and incubated overnight at 4°C with primary antibodies against Sema3A (1:1000; Abcam, Cambridge, MA, United States) and phosphorylated (p-)GSK3β, GSK3β, p-CRMP2, CRMP2, and β-actin (all 1:1000 and from Cell Signaling Technology, Danvers, MA, United States). The membrane was then incubated with horseradish peroxidase-conjugated secondary antibody (1:1000; Cell Signaling Technology, Danvers, MA, United States) for 1 h at room temperature and immunoreactivity was analyzed using ImageJ.
7
Western Blot Analysis of Cellular Proteins
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For Western blot analysis, cells were lysed using lysis buffer containing 10 mM Tris/HCl (PH 7.5), 150 mM NaCl, 2 mM EDTA, 1% (v/v) Triton X-100, 1 mM Na3CO4, 1 mM PMSF, and 0.1 mM aprotinin. The cells were scraped from the plates and centrifuged at 16,060 g for 30 min at 4°C. For nuclear protein extraction, the pellet from the 700 g centrifugation was washed by buffer A, resuspended in buffer B containing 20 mM HEPES, pH 7.9, 1.5 mM MgCl2, 420 mM NaCl, 0.2 mM EDTA, 10 mM NaF, 2 mM Na3VO4, 1 mM pyrophosphoric acid, and Complete TM protease inhibitors (Cell Signaling, USA) and incubated on ice for 5 min. The protein concentration in the cell lysates was determined using the Bradford protein assay. Western blot analysis was performed as previously described using the several antibodies (Bennett et al., 2005 (link)). Sema3A, plexin A1, and NRP1 antibodies were obtained from ABcam, Cambridge, UK. PLCγ2, phospho-PLCγ2, β-cateinin, and DAP12 antibodies were from Cell Signaling, USA.
8
Immunofluorescence Analysis of Cell Markers
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Paraffin sections, dewaxing and rehydration of paraffin sections, antigen repair, blocking endogenous peroxidase, BSA blocking, and primary antibody incubation were performed as previously mentioned. For cell creep, cells were cleaned with PBS and fixed at room temperature for 20–30 min using 4% paraformaldehyde. Further washing with PBS and permeabilizing the film with 0.1%–0.5% Triton X‐100 was conducted for 15–20 min. Cells/Tissues were blocked with 5% BSA and then incubated with primary antibodies Nrp1 (Abcam, The United States), Sema3A (Abcam, The United States), ALP (Servicebio, China) at 4°C overnight. After washing with PBS, fluorescent secondary antibodies were added to the tissue and incubated for 1 h at room temperature while shielding from light. Further washing with PBS and DAPI dye staining for 3–5 min was carried out. After additional washing with PBS, anti‐fluorescence quenching tablet seals were performed.
9
Comprehensive Protein Extraction and Analysis
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Total protein was extracted with lysis buffer (Beyotime, Shanghai, China) containing protease inhibitor cocktail. Cytoplasmic/nuclear proteins were extracted with nuclear and cytoplasmic extraction reagents (KeygenBiotech, Nanjing, China). Proteins were quantified with Coomassie Brilliant Blue, and BSA served as the standard. Proteins were separated by SDS-PAGE on a 10% gel and transferred onto PVDF membranes (Millipore, MA, USA). The membranes were blocked with 5% skim milk at room temperature for 2 h and then incubated with primary antibodies for SEMA3A, NRP1, active-caspase-3, Plexin A1, A2, A3, A4, D1 (Abcam, USA), P65, p-P65, IκB, p-IκB, CDK2/4/6, cyclin E1, cyclin D1/D3, P21, P27, caspase-7 (CST, USA), E-cadherin, N-cadherin, Vimentin, β-catenin, β-actin, H1, cleaved-caspase-5, and SNAI2 (Bioworld, China). All antibodies were used at a 1:1000 dilution. The membranes were incubated with anti-goat IgG HRP-conjugated secondary antibodies (ZhongshanGoldenbridge Bio) for 1 h at room temperature. Immunoreactive bands were detected with Immobilon Western Chemiluminescent HRP Substrate (Millipore) and visualized using an ImageQuantLAS 4000 mini imaging system (General Electric). Analyses of the bands were performed using ImageJ software.
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