Animals were sacrificed by decapitation and the L4/5 spinal dorsal horns were harvested and temporarily stored. Then, the samples were homogenized in ice‐cold RIPA lysis buffer. After measurement of concentration, the protein samples were separated on 10% SDS‐PAGE and electro‐transferred onto PVDF membranes. Protein samples were then incubated with antibodies for rabbit anti‐CXCL12 (1:1000; Abcam), rabbit anti‐CXCR4 (1:1000, Abcam), and mouse anti‐Actin (1:2000; Santa). The membranes were incubated with horseradish peroxidase–conjugated anti‐mouse or anti‐rabbit secondary antibodies (1:2000, Jackson), and exposed to film. The intensity of the selected bands was analyzed using Image J software.
Rabbit anti cxcl12
Manufactured by Abcam
Sourced in Italy, United States
Rabbit anti-CXCL12 is a primary antibody that recognizes the CXCL12 protein. CXCL12, also known as stromal cell-derived factor 1 (SDF-1), is a chemokine that plays a role in cellular processes such as cell migration and adhesion. This antibody can be used for the detection and quantification of CXCL12 in various applications.
Automatically generated - may contain errors
Lab products found in correlation
Rabbit anti cxcr4, by Abcam (3 mentions)
Rabbit anti osterix, by Santa Cruz Biotechnology (2 mentions)
Horseradish peroxidase conjugated anti rabbit or anti mouse secondary antibodies, by Jackson ImmunoResearch (1 mentions)
Biotinylated goat anti mouse igg, by Vector Laboratories (1 mentions)
Dm2500 microscope, by Leica (1 mentions)
Mouse anti nestin, by Abcam (1 mentions)
Mouse anti tlr4, by Abcam (1 mentions)
Mouse anti gapdh, by Abcam (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Enhanced chemiluminescence kit, by Beyotime (1 mentions)
West pico chemiluminescence substrate, by Thermo Fisher Scientific (1 mentions)
Glass coverslip, by Avantor (1 mentions)
Goat anti rabbit igg fitc, by Merck Group (1 mentions)
Vectashield mounting medium with dapi, by Vector Laboratories (1 mentions)
Rat anti mouse cd31, by BD (1 mentions)
Cell lysis buffer, by Cell Signaling Technology (1 mentions)
Rabbit anti traf6, by Abcam (1 mentions)
Liteablot turbo luminol reagents, by Euroclone (1 mentions)
Hyperfilm ecl film, by Euroclone (1 mentions)
6 protocols using rabbit anti cxcl12
1
Western Blot Analysis of CXCL12/CXCR4 Signaling
2
Femur Histological Analysis Protocol
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Femurs, dissected from adhering tissues, were fixed in 4% PFA and decalcified as previously described [13 (link)]. Samples were embedded with Tissue-Tek OCT compound. Then, 12 μm thick sections of femurs were obtained by a rotatory -30°C air-dried microtome cryostat (Ames Cryostat Miles). Sections were stained with toluidine blue or with hematoxylin and eosin (H&E) stains.
Other sections, after rinsing with PBS and incubation with 0,3% H2O2 were incubated with blocking buffer (0.3% Triton X-100 and 1% BSA in PBS) containing 10% normal serum for 30-60 min at RT in a humidified chamber. Then, sections were incubated 1 h a RT with the following primary antibodies diluted 1:80 in blocking buffer: rabbit anti-Osterix (Santa Cruz Biotechnology, DBA, Milano, Italy); rabbit anti-CXCL12 (Abcam; Prodotti Gianni, Milano, Italy); mouse anti-nestin (Abcam; Prodotti Gianni, Milano, Italy). After washing in PBS, sections were incubated for 30 min at RT with a biotinylated goat anti-mouse IgG (Vector Laboratories, DBA Milano, Italy) or with a biotinylated goat anti-rabbit IgG (Bethyl Laboratories, Aurogene s.r.l., Roma, Italy) both diluted 1:200 in blocking buffer. Control experiments were performed by omitting the primary antibody. Slides were imaged using a Leica DM 2500 microscope.
Other sections, after rinsing with PBS and incubation with 0,3% H2O2 were incubated with blocking buffer (0.3% Triton X-100 and 1% BSA in PBS) containing 10% normal serum for 30-60 min at RT in a humidified chamber. Then, sections were incubated 1 h a RT with the following primary antibodies diluted 1:80 in blocking buffer: rabbit anti-Osterix (Santa Cruz Biotechnology, DBA, Milano, Italy); rabbit anti-CXCL12 (Abcam; Prodotti Gianni, Milano, Italy); mouse anti-nestin (Abcam; Prodotti Gianni, Milano, Italy). After washing in PBS, sections were incubated for 30 min at RT with a biotinylated goat anti-mouse IgG (Vector Laboratories, DBA Milano, Italy) or with a biotinylated goat anti-rabbit IgG (Bethyl Laboratories, Aurogene s.r.l., Roma, Italy) both diluted 1:200 in blocking buffer. Control experiments were performed by omitting the primary antibody. Slides were imaged using a Leica DM 2500 microscope.
3
Protein Expression Analysis of Spinal Cord
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After the behavioral tests were complete, the rats were anesthetized with an overdose of pentobarbital, and the L4–6 segments of the spinal cord were removed and processed for further biological analysis. After the samples were homogenized, cytoplasmic extracts were purified from each specimen using nucleoprotein and cytoplasmic protein extraction kits according to the manufacturer’s instructions (KGP-150; KangChen, Shanghai, China). Equivalent protein samples (50 μg) were separated using 10% SDS-PAGE, and the proteins were then transferred onto PVDF membranes (EMD Millipore, USA). The primary antibodies used in this experiment were rabbit anti-CXCL12 (1:300; Abcam, USA), rabbit anti-CXCR4 (1:200; Abcam, USA), mouse anti-TLR4 (Abcam) and anti-mouse GAPDH (dilution 1:10,000, Abcam). The membranes were incubated with the primary antibodies overnight on a shaker at 4°C. Then, the proteins were detected with horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies (BioSS, Beijing, China) for 1 h and visualized using an enhanced chemiluminescence kit (Beyotime Biotechnology, China). Semi-quantitation of the scanned images was performed using Quantity One software (Bio-Rad Laboratories, Milan, Italy).
4
Western Blot Analysis of CXCL12 and CXCR4
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The DRG of CCD mice (n = 4) were combined as one sample for western blot, the DRG of control mice (n = 4) were combined as the same way. Briefly, L4 and L5 lumbar DRG were dissected in control mice and CCD mice and placed temporarily in liquid nitrogen. Then the samples were homogenized in ice-cold lysis buffer by ultrasonic homogenizer (Cole parmer instruments, USA). The crude homogenates were centrifuged at 4 °C for 15 min at 3 000 rpm, and the supernatants were collected. After the protein concentrations were determined, the samples were heated for 5 min at 99 °C, and 30–60 μg protein was loaded onto 12% SDS–polyacrylamide gels, then electrophoretically transferred onto PVDF membranes. The membranes were blocked with 3% non-fat milk for 1 h and incubated overnight at 4 °C with primary antibody. The following primary antibodies were used: rabbit anti-CXCL12 (1:200, Abcam), rabbit anti-CXCR4 (1:200, Abcam), and mouse anti-β-actin (1:1000, CST). The proteins were detected with horseradish peroxidase-conjugated anti-rabbit secondary antibodies (1:1000, CST), visualized using the supersignal west pico chemiluminescence substrate (Thermo. USA), and exposed in Bio-rad chemiDox-XRS imagine system.
5
Immunofluorescence Analysis of CD34+ Cells
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CD34+ cells were spread on a glass coverslip (VWR International, Leuven, Belgium) and fixed with cold methanol for 10 min at −20°C, before incubation with specific antibodies at room temperature for 60 min. Cells were then incubated at room temperature for 60 min with following antibodies: mouse anti-human α-SMA conjugated with Cy3 (Sigma-Aldrich, St. Louis, MO, USA), rat anti-mouse CD31 (BD) and rabbit anti-collagen-1, rabbit anti-angiopoietin-1, rabbit anti-angiopoietin-2, rabbit anti-TIE-2, and rabbit anti-CXCL12 (all from Abcam). Second layer staining was with a goat anti-rat IgG-FITC, a goat anti-rabbit IgG-FITC, and a rabbit anti-goat IgG-FITC (all from Sigma-Aldrich). Stained cells were mounted in Vectashield mounting medium with DAPI (Vector Laboratories), and captured by a Leica DMIRBE confocal microscope (Leica, Wetzlar, Germany) as above.
6
Western Blot Analysis of Bone Cells
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Proteins from total BMCs and from BMSCs were extracted in cell lysis buffer (Cell Signaling, EuroClone) after 2 days of culture, and the concentration was determined by the BCA protein assay reagent (Pierce, EuroClone). Western blotting was performed as previously described (Sabbieti et al. 2010) (link). Membranes were immunoblotted in blocking buffer with specific antibodies: rabbit anti-TNFα and rabbit anti-NF-κB (BioLegend, Microtech SrL, Napoli, Italy) both diluted 1:500; mouse anti-RANKL, rabbit anti-TRAF6, rabbit anti-CXCL12 and rabbit anti-TGFβ (Abcam, Prodotti Gianni) all diluted 1:600; rabbit anti-PPRγ (Santa Cruz Biotechnology, Inc. DBA) diluted 1:300 and rabbit anti-Osterix (Santa Cruz Biotechnology, DBA) diluted 1:300. After washing with PBS-T, blots were incubated with horseradish peroxidase (HRP)-conjugated donkey anti-rabbit IgG or with HRP-conjugated rabbit anti-mouse IgG (Cell Signaling, EuroClone) both diluted 1:50,000. Immunoreactive bands were visualized using LiteAblot Turbo luminol reagents (EuroClone) and Hyperfilm-ECL film (EuroClone) according to the manufacturer's instructions. To normalize the bands, filters were stripped and re-probed with a monoclonal anti-α-tubulin (Sigma-Aldrich). Band density was quantified densitometrically.
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