For western blotting, mouse anti-p63 (4A4) (LabVision, Kalamazoo, MI, USA; 1/1000 dilution), mouse anti-β-actin (1/1000 dilution), rabbit anti-SDF1 (1/1500 dilution) (Cell Signaling, Boston, MA, USA), rabbit anti-CXCR4 (1/500) (Abcam, Cambridge, MA, USA) antibodies were used. For immunofluorescence experiments, rabbit anti-CXCR4 (1/500) (Abcam, Cambridge, MA, USA) (Figure 1C ) or mouse anti-CXCR4 (R&D Systems, Minneapolis, MN, USA; 8μg/ml dilution) (Supplemental Figure 2B ) antibodies were used. Secondary antibodies used were anti-mouse and anti-rat IgG horseradish peroxidase-conjugated for immunoblot and goat anti-rabbit-alexafluor-555 or goat anti-mouse-alexafluor-488 conjugated antibodies for immunofluorescence.
Rabbit anti cxcr4
Manufactured by Abcam
Sourced in United States, United Kingdom
Rabbit anti-CXCR4 is a primary antibody that recognizes the CXCR4 protein, a chemokine receptor expressed on the surface of various cell types. This antibody can be used for the detection and study of CXCR4 in research applications.
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Lab products found in correlation
Rabbit anti cxcl12, by Abcam (3 mentions)
Mouse anti β actin, by Cell Signaling Technology (1 mentions)
Mouse anti cxcr4, by R&D Systems (1 mentions)
Ketamine, by Zoetis (1 mentions)
Horseradish peroxidase conjugated anti rabbit or anti mouse secondary antibodies, by Jackson ImmunoResearch (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Rabbit anti tnf α, by Abcam (1 mentions)
Hrp linked anti rabbit igg, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Bradford assay kit, by Bio-Rad (1 mentions)
X ray film, by Kodak (1 mentions)
Enhanced chemi luminescence (ecl), by Bio-Rad (1 mentions)
Cell lysis buffer, by Cell Signaling Technology (1 mentions)
Liteablot turbo luminol reagents, by Euroclone (1 mentions)
Hyperfilm ecl film, by Euroclone (1 mentions)
Lsm 510 confocal microscopy, by Zeiss (1 mentions)
Mouse anti α sma, by Merck Group (1 mentions)
Alexa fluor 546, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Boster Bio (1 mentions)
13 protocols using rabbit anti cxcr4
1
Western Blot and Immunofluorescence Antibody Conditions
2
Quantifying Angiogenesis in Ischemic Limbs
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Whole ischemic limbs were harvested, the adhering tissues and femora were carefully removed, and the samples were immersion-fixed with 4% buffered paraformaldehyde. The staining was performed on serial 5-μm-thick paraffin-embedded sections. Immunohistochemical staining was performed on mouse ischemic thigh muscles (sartorius muscle, gracilis muscle, adductor muscle and semimembranosus muscle were included) using goat anti-Von Willebrand factor (vWF; Santa Cruz Biotechnologies, San Jose, CA, USA), rabbit anti-CD34 (Millipore, Billerica, MA, USA), and rabbit anti-CXCR4 (Abcam, San Francisco, CA, USA) antibodies, followed by counterstaining with Hoechst. The stained slides were observed using fluorescence microscopy. Three cross-sections were analyzed for each animal. Ten different fields from each tissue preparation were randomly selected, and visible capillaries were counted. The densities of capillaries and CXCR4 expression were expressed as the fluorescence/myofiber ratio.
3
Measuring CXCR4 in Rat Hypothalamus
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Rats in the third group, after being given access for 5 days to a HFD or chow diet, were deeply anesthetized with 20 mg/kg xylazine (LLOYD Incorporated, Shenandoah, IA, USA) and 100 mg/kg ketamine (Fort Dodge Animal Health, Overland Park, KS, USA) (i.p.). They were perfused transcardially with 0.9% NaCl and then 4% paraformaldehyde, as previously described (Barson et al., 2015 (link)). Brains were removed, post-fixed in 4% paraformaldehyde for 24 h, transferred to 25% sucrose for 4 days at 4°C, and then frozen at -80°C. Coronal brain slices containing the different regions of the hypothalamus were processed for CXCR4 immunohistochemistry, using a 1:200 dilution of rabbit anti-CXCR4 (Abcam, Cambridge, MA, USA) and a 1:400 dilution of Alexafluor secondary antibody, goat anti-rabbit 594 (Life Technologies, Grand Island, NY, USA). Several antibodies for CXCR7 were tested but did not label properly, suggesting a lack of effective commercially available antibodies for CXCR7. Although protein levels of CXCR7 could not be measured, the existence of high levels of CXCR7 mRNA suggests a possibility that this receptor may be present in these brain regions as shown in transgenic mice (Banisadr et al., 2003 (link)).
4
Western Blot Analysis of CXCL12/CXCR4 Signaling
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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.
5
Western Blot Analysis of CXCR4 and TNF-α
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Tissue samples of spinal cord were homogenized (Polytron, Kinematica, Switzerland) in 100μL ice-cold lysis buffer with protease inhibitor cocktails (Sigma-Aldrich, USA). Protein concentrations were determined by the Bradford assay kit (Bio-Rad, USA). Equal amount of proteins was subjected to electrophoresis in 12.5% SDS-polyacrylamide gels and then transferred to polyvinylidene fluoride (PVDF) membranes (Bio-Rad, USA). After blocking with 5% nonfat milk for 2 h, the membranes were incubated with rabbit anti-CXCR4 (1 : 1000, Abcam, UK), rabbit anti-TNF-α (1 : 1000, Abcam, UK), and β-Tubblin (1 : 1000, Cell Signaling, USA) overnight at 4°C. After washing with 0.01 M Tris-buffered saline Tween-20 for 10 min 3 times, the membranes were incubated for 2 h at room temperature with secondary antibodies HRP-linked anti-rabbit IgG (1 : 2000, Cell Signaling, USA). Protein expressions were visualized by exposure to X-ray films (Kodak, USA) after incubation of blot in ECL (Bio-Rad, USA) and quantified by ImageJ software (National Institutes of Health, USA).
6
Quantifying CXCL12 and CXCR4 Protein Levels
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Proteins from BMCs were extracted in cell lysis buffer (Cell Signaling, EuroClone, Milano, Italy) after 3 days of culture, and the concentration was determined by the BCA protein assay reagent (Pierce, EuroClone). Western blotting was performed as previously described [14 (link)]. Membranes were immunoblotted in blocking buffer with rabbit anti-CXCL12 or with rabbit anti-CXCR4 (Abcam, Prodotti Gianni, Milano, Italy) both diluted 1:600. After washing with PBS-T, blots were incubated with horseradish peroxidase (HRP)-conjugated donkey anti-rabbit IgG IgG (Cell Signaling, EuroClone) diluted 1:100,000.
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.
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.
7
HA-Lm Gel Modulates CXCR4 Expression
8
Immunohistochemical Analysis of Brain Sections
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Brain sections (thickness 30 μm) from 4% paraformaldehyde-perfused animals were used. After blocking in 5% NDS in Tx/PBS for 60 minutes, sections were incubated with primary antibody in Tx/PBS in 5% NDS at 4°C overnight. The following antibodies were used: rat anti-cluster of differentiation 68 (CD68; diluted at 1:200; AbD Serotec, Düsseldorf, Germany), rabbit anti-CXCR4 (diluted at 1:200; Abcam), mouse anti-CD45 (diluted at 1:25; AbD Serotec) and rabbit anti-ionized calcium binding adaptor molecule 1 (Iba1; diluted 1:2000; Wako Chemicals, Neuss, Germany). The next day, the sections were rinsed with 1% NDS in Tx/PBS three times for 10 minutes. All the following steps were performed in the dark in order to preserve the fluorophores conjugated to the secondary antibodies. Sections were incubated with secondary antibodies in 2% NDS in Tx/PBS for 90 minutes at room temperature and rinsed in PBS three times for 10 minutes. Finally, the sections were rinsed in PBS and mounted on supercharged glass slides, allowed to dry, cover-slipped using PVA-DABCO and analyzed using LSM 510 confocal microscopy (Carl Zeiss).
9
Immunostaining of Vascular Cell Markers
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Immunostaining was performed on carotid or mouse femoral artery sections following our published method 21 . Briefly, the sections were first incubated with each of the primary antibodies for 12 hour with dilution ratios as follows: rabbit anti‐CXCR4 (Abcam, Cambridge, MA), 1:200; rabbit anti‐SDF‐1α (Santa Cruz Biotechnology Inc., Dallas, TX), 1:200; mouse anti‐α‐SMA (Sigma‐Aldrich, St. Louis, MO), 1:1000. Normal IgG was used for background control. The detected proteins were then visualized by fluorescence microscopy after incubating the cells with species‐matched secondary antibodies conjugated with either Alexa Fluor 546 or Alexa Fluor 488 (Invitrogen, Carlsbad, CA).
For quantification, five immunostained sections from each animal were used. Positively stained cells were counted (based on fluorescence intensity) using ImageJ by a student blinded to experimental conditions. The numbers from all five sections were pooled to generate the mean for each animal. The means from all the animals in each treatment group were then averaged, and the SEM was calculated.
For quantification, five immunostained sections from each animal were used. Positively stained cells were counted (based on fluorescence intensity) using ImageJ by a student blinded to experimental conditions. The numbers from all five sections were pooled to generate the mean for each animal. The means from all the animals in each treatment group were then averaged, and the SEM was calculated.
10
Colocalization of CXCR4 and CD74 in Endothelial Cells
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Endothelial cells were cultured on cover slips in 24-well plates at
a concentration 4 × 104 cells per well for 24 h at
37 °C. Next, the cells were infected with P.
gingivalis at an MOI of 100 for 24 h. The cells were subsequently
washed and fixed, after which they were incubated in 1% BSA in 0.1% PBS-Tween
for 1 h. Afterwards, the samples were incubated with mouse anti-CD74 (1:100;
Santa Cruz) and rabbit anti-CXCR4 (1:200; Abcam) overnight at 4 °C. Donkey
anti-mouse IgG labeled with PE (1:20; Proteintech) and goat anti-rabbit IgG
labeled with FITC (1:20; Proteintech) were used as the secondary antibodies and
were incubated with the cells for 1 h at room temperature. DAPI (Boster, Wuhan,
China) was used to stain the cell nucleus (pseudo-colored blue), and the cells
were observed using a fluorescence microscope (Nikon 80i, Japan) to evaluate the
colocalization between CXCR4 and CD74.
a concentration 4 × 104 cells per well for 24 h at
37 °C. Next, the cells were infected with P.
gingivalis at an MOI of 100 for 24 h. The cells were subsequently
washed and fixed, after which they were incubated in 1% BSA in 0.1% PBS-Tween
for 1 h. Afterwards, the samples were incubated with mouse anti-CD74 (1:100;
Santa Cruz) and rabbit anti-CXCR4 (1:200; Abcam) overnight at 4 °C. Donkey
anti-mouse IgG labeled with PE (1:20; Proteintech) and goat anti-rabbit IgG
labeled with FITC (1:20; Proteintech) were used as the secondary antibodies and
were incubated with the cells for 1 h at room temperature. DAPI (Boster, Wuhan,
China) was used to stain the cell nucleus (pseudo-colored blue), and the cells
were observed using a fluorescence microscope (Nikon 80i, Japan) to evaluate the
colocalization between CXCR4 and CD74.
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