CXCR4 protein production was determined by western blotting. Total protein was isolated utilizing radioimmunoprecipitation assay buffer (Thermo Scientific). Proteins were separated via gel electrophoresis on NuPage Novex Bis-Tris Mini Gels (Invitrogen) at 200 V for 35 min. Separated proteins were transferred to a polyvinylidene difluoride membrane (Invitrogen) via the XCell II Blot Module (Invitrogen) at 30 V for 1 h. To assess CXCR4 expression, blotted membranes were stained for anti-human CXCR4 (product ab124824, 1:100 dilution, Abcam) utilizing a horseradish peroxidase-labeled secondary antibody (product ab6721, 1:10000 dilution, Abcam). Staining with anti-β actin (product ab8227, 1:2000 dilution, Abcam) served as a loading control. X-ray films were developed with the chemiluminescent reagent SuperSignal West Pico (Thermo Scientific). Three replicates were performed per assay.
Ab124824
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab124824 is a primary antibody that targets a specific protein. It is suitable for use in various research applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab6721, by Abcam (4 mentions)
Ab9797, by Abcam (4 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Ab8227, by Abcam (3 mentions)
Ab25117, by Abcam (3 mentions)
Autostainer link 48, by Agilent Technologies (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Ab39012, by Abcam (2 mentions)
Ab15580, by Abcam (2 mentions)
Ab38898, by Abcam (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
A1r confocal microscope, by Nikon (2 mentions)
Nupage novex bis tris mini gel, by Thermo Fisher Scientific (1 mentions)
Xcell 2 blot module, by Thermo Fisher Scientific (1 mentions)
Radioimmunoprecipitation assay buffer, by Thermo Fisher Scientific (1 mentions)
Supersignal west pico, by Thermo Fisher Scientific (1 mentions)
Polyvinylidene difluoride membrane, by Thermo Fisher Scientific (1 mentions)
Picrosirius red solution, by Merck Group (1 mentions)
Evos fluorescence microscope, by Thermo Fisher Scientific (1 mentions)
59 protocols using ab124824
1
CXCR4 Protein Expression Analysis
2
Immunohistochemical Staining and Quantification
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Tissue slides were stained as previously described on the paraffin‐embedded slides (Damhofer et al., 2015 ). Antibodies and dilutions used were CK19 1 : 500 (MU246‐UC; Biogenex), CXCR4 1 : 400 (Ab124824; Abcam, Cambridge, UK), Ki67 1 : 2000 (SAB5500134; Sigma). For picrosirius red staining, slides were deparaffinized, stained in a 0.1% picrosirius red solution (Sigma) in saturated picric acid for 1 h, and washed three times with 0.1 m acetic acid solution. All slides were imaged on an Olympus BX51 (Tokyo, Japan). Quantification of Ki67 staining was performed with Fiji count particles (Schindelin et al., 2012 ), after DAB/H color deconvolution. For IF images, slides were cut at 10 μm, mounted in Prolong Gold (ThermoFisher), and imaged on an EVOS fluorescence microscope (ThermoFisher). For collagen staining in tissue culture vessels, cells were cultured as described and after 7 days of coculture washed with PBS three times prior to fixation in 4% paraformaldehyde for 15 min. Following three washes with PBS, cells were stained for their collagen deposition as described above for 18 h and were equally treated as the tissue slides. Cells were subsequently imaged on an Olympus BX51. Quantifications of the percentage of Ki67‐positive nuclei, width of HE staining, or percentage of Venus‐positive cells were performed using Fiji package of imagej .
3
Western Blot Analysis of Cellular Proteins
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Tissue or cellular protein was extracted with 1 × cell lysis buffer (Promega, Madison, WI). Western blot was performed as previously described [9 (link), 11 (link)–15 (link)], with antibodies for HNF4A (ab181604), hexokinase 2 (HK2, ab104836), solute carrier family 2 member 1 (SLC2A1, ab40084), v-myc avian myelocytomatosis viral oncogene neuroblastoma-derived homolog (MYCN, ab16898), hnRNPU (ab10297), CTCF (ab188408), clusterin (CLU, ab69644), C-X-C motif chemokine receptor 4 (CXCR4, ab124824), trophoblast glycoprotein (TPBG, ab129058), uveal autoantigen with coiled-coil domains and ankyrin repeats (UACA, ab99323), FLAG (ab125243), Myc (ab9106), glutathione S-transferase (GST, ab19256), histone H3 (ab5103), or β-actin (ab6276, Abcam Inc., Cambridge, MA).
4
Quantitative Analysis of Pluripotency and Stem Cell Markers
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The protein expression was determined by western blot analysis. In brief, the sample cells were lysed by radio-immunoprecipitation assay buffer (RIPA; Roche, Basel, Switzerland) containing protease and phosphatase inhibitors, then resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and blotted onto 0.45 μm polyvinylidene fluoride membranes (Merck Millipore, Burlington, MA, USA). The membranes were subsequently blocked in 5% BSA and probed with primary antibodies: anti-Nanog (#4903; Cell Signaling Technology), anti-Oct4 (#2750S; Cell Signaling Technology), anti-Sox2 (#14962S; Cell Signaling Technology), anti-CXCR4 (ab124824; Abcam), and anti-Glyceraldehyde 3-phosphate dehydrogenase (GAPDH, ab181603; Abcam) at 4 °C overnight. After extensive washing, the membranes were incubated with horseradish peroxide-conjugated secondary antibodies (HRP-conjugated 2nd antibodies): goat anti-rabbit IgG (ab6721; Abcam) and goat anti-mouse IgG (ab6789; Abcam) for 1 h at room temperature, then developed using enhanced chemiluminescence (ECL; Merck Millipore). Immuno-reactive bands were detected by UVP BioSpectrum 810 imaging system (Thermo Fisher Scientific) and analyzed by VisionWorks LS software (UVP, CA, USA). The protein expression levels were normalized to GAPDH as an internal loading control.
5
Immunocytochemical Staining of ASCs
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For immunocytochemical (ICC) staining, the harvested ASCs from each cycle were replated onto TCPS for 24 h. Afterwards, cells were fixed in 4% paraformaldehyde for 30 min at 4 °C and blocked in 3% BSA for 30 min at room temperature. The primary antibody against CXCR4 (ab124824; Abcam) was incubated overnight at 4 °C. After PBS washing, cells were incubated with the Cy3-conjugated goat anti-rabbit secondary antibody (Jackson ImmunoResearch Laboratories) and counterstained with Phalloidin (ab176756; Abcam) and DAPI (Santa Cruz Biotechnology) for 1 h at room temperature in the dark. These immuno-stained cells were captured using a fluorescent microscope (Leica).
6
Quantifying AML Organ Infiltration
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IHC analysis was performed in paraffin-embedded tissue samples using CD45 antibodies (IR75161-2, Agilent Technologies) and CXCR4 antibodies (ab124824, Abcam). IHC staining was performed in an Autostainer Link 48 (Agilent Technologies), following the manufacturer’s instructions. AML organ infiltration by CD45 and CXCR4 expression was evaluated and quantified using an Olympus BX53 microscope and Image J Fiji software v.1.8.0.172 (NIH), choosing six fields randomly for each tissue. The results were expressed as stained total area ± SEM.
7
Profiling CXCR4 Expression in BMSC Subsets
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The CXCR4 expression profiles in the two types of BMSCs at two passages (P3 and P6) were assessed by immunofluorescence staining. Cells were collected and fixed in 4% paraformaldehyde at 4°C for 20 min. Following treatment with 0.5% Triton X-100 and 1% bovine serum albumin (Thermo Fisher Scientific, Inc.) for 1 h at 25°C, cells were incubated with rabbit anti-CXCR4 antibody (ab124824; 1:100; Abcam, Cambridge, MA, USA) overnight at 4°C. After washing with PBS, cells were incubated with Alexa Fluor® 488-conjugated anti-CXCR4 antibody (ab208128; Abcam) for 30 min at 37°C followed by DAPI (Sigma-Aldrich; Merck KGaA) staining for 2 min, to counterstain the cell nuclei. The sections were mounted and then observed under a Zeiss LSM 710 confocal microscope (Carl Zeiss AG, Oberkochen, Germany) and the intensity of the CXCR4-positive area were measured using Image-Pro Plus 6.0 software. The surface expression of CXCR4 was also analyzed using flow cytometric analysis as previously described (22 (link)). The cells were incubated with FITC-conjugated anti-CXCR4 antibody (Abcam).
8
Immunohistochemical Assessment of CXCR4, CD Markers, and TLR9 in LSG Biopsies
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Immunohistochemical staining to detect C-X-C chemokine receptor type 4 (CXCR4), CD19, CD21, Toll-like receptor 9 (TLR9) and intercellular cell adhesion molecule 1 (ICAM1) was performed on LSG biopsy sections as described previously [25 (link)]. CXCR4 (ab124824, Abcam, Cambridge, MA, USA), CD3 (ab699, Abcam, Cambridge, MA, USA), CD19 (ab134114, Abcam, Cambridge, MA, USA), CD21 (ab75985, Abcam Cambridge, MA, USA), ICAM1 (ab53013, Abcam, Cambridge, MA, USA), CD20 (ab78237, Abcam, Cambridge, MA, USA), and TLR9 (BA3861-1, Boster Biotechnology, Wuhan, China) antibodies were used in this experiment. Negative control staining was performed by replacing primary antibodies with PBS. Positive immunoreactivity appeared as a brown color. Double staining for CD3 and CD20 was used to analyze T/B cell segregation using the DouMaxvision™ double-stain system (KIT-9998, Maixin Biotechnology, Fuzhou, China). A scoring system was used to describe the results of double staining as described previously [26 (link)]. The images of grade 1 to 3 are shown in Additional file 2 : Figure S3.
9
CXCR4-HIF-1α Interaction Visualization
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ACHN cells were seeded on tissue culture-treated chamber slides (REF 354108; BD Falcon, BD Biosciences, BD AB, Stockholm, Sweden). The following day, the cells (1×106) were serum-starved for 12 h prior to 24-h treatment with CXCL12α (200 ng/ml). The cells were then fixed as described previously [16 (link)], and the slides were blocked in blocking solution (Sigma) for 30 min at 37 °C. After washing, the slides were incubated with Duolink PLA Rabbit MINUS and PLA Mouse PLUS proximity probes (Olink Bioscience, Uppsala, Sweden), and proximity ligation was performed using the Duolink detection reagent kit (Olink Bioscience) according to the manufacturer’s protocol. Fluorescence was detected using an LSM-510 laser scanning microscope (Carl Zeiss, Thornwood, NY). The antibodies used for the PLA were rabbit anti-CXCR4 (ab124824; Abcam) and mouse anti-HIF-1α (ab1, Abcam) antibodies.
10
Western Blot Analysis of Inflammatory Proteins
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Protein (20–50 μg) was extracted from the ipsilateral spinal cord dorsal horn and separated with 10% SDS-PAGE gel, transferred onto a nitrocellulose membrane, and incubated with antibody against CXCR4 (1:1000; ab124824, Abcam), VDUP-1 (1:1000; sc-271238, Santa Cruz Biotechnology), NLRP3 (1:500; NBP1-77080, Novus Biologicals), apoptosis-associated speck-like molecule containing CARD domain (ASC; 1:1000; sc-22514-R, Santa Cruz Biotechnology), Caspase1 (1:1000; ab-1872, Abcam), Caspase1 p10 (1:1000; sc-514, Santa Cruz Biotechnology), and IL-1β (1:1000; ab9722, Abcam) or control β-Actin (1:5000; YM3028, ImmunoWay). Blots were washed and incubated in HRP-linked anti-rabbit IgG antibody (1:5000; 7074, Cell Signaling Technology) and HRP-linked anti-mouse IgG antibody (1:5000; 7076, Cell Signaling Technology). Protein blots were visualized using Clarity ECL Substrates (Biorad).
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