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Ab8021

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Ab8021 is a laboratory equipment product. It serves a core function in scientific research and analysis.

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Lab products found in correlation

Ab25088, by Abcam (6 mentions) Ab32144, by Abcam (4 mentions) Ab5076, by Abcam (3 mentions) Phosphor akt s473, by Cell Signaling Technology (2 mentions) T8203, by Merck Group (2 mentions) Mca497rt, by Abcam (2 mentions) Ab14041, by Merck Group (2 mentions) Ab5076, by Bio-Rad (2 mentions) Ab39626, by Santa Cruz Biotechnology (2 mentions) Ab5603, by Santa Cruz Biotechnology (2 mentions) Sc 17320, by R&D Systems (2 mentions) Af2418, by Merck Group (2 mentions) Mca711gt, by Abcam (2 mentions) Sc 33560, by Bio-Rad (2 mentions) M082301, by Agilent Technologies (2 mentions) Abc kit, by Vector Laboratories (2 mentions) Dab kit, by Vector Laboratories (2 mentions) Ab15580, by Abcam (2 mentions) Ab14041, by Abcam (2 mentions) Ba1054 ba1050, by Boster Bio (2 mentions)

23 protocols using ab8021

1

Immunofluorescent Detection of CX3CL1 and CX3CR1

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Rat carotid arteries were harvested 24 h after injury and stained for CX3CL1 to determine presence of the protein. Primary antibody (rabbit polyclonal to CX3CL1, Abcam: ab25088) and secondary antibody (goat anti-rabbit IgG highly cross-adsorbed Alexa 647) dilutions were 1:200 and 1:3000, respectively. Aortic roots of mice fed a high-fat diet for 16 weeks were stained for CX3CR1 to determine presence of the protein in the atherosclerotic plaque. Primary antibody (rabbit polyclonal to CX3CR1, Abcam: ab8021) and secondary antibody (goat anti-rabbit IgG Alexa 555, Invitrogen or Alexa 647 highly cross-adsorbed, Invitrogen A32733) dilutions were 1:200 and 1:3000, respectively. Quantification of CX3CL1 staining data are presented as number of fluorescent pixels, which are the average of 5 images per animal, n = 3 animals per treatment group. Fluorescent imaging of CX3CR1 in arteries from atherosclerotic mice was obtained but not quantified due to low sample numbers. Results are expressed as mean number of fluorescent pixels ± the standard error of the mean (SEM).
Tissue sections from LDL KO mice were stained for CX3CR1 (Abcam ab8021) using primary dilution of 1:200 and secondary goat anti-rabbit Alexa 555 dilution of 1:3000 (Life Technologies A21425). Nuclear staining was carried out using DAPI at 1:500 dilution. Digital images were acquired as mentioned above.
Kassam H.A., Gillis D.C., Dandurand B.R., Karver M.R., Tsihlis N.D., Stupp S.I, & Kibbe M.R. (2020). Development of Fractalkine-Targeted Nanofibers that Localize to Sites of Arterial Injury. Nanomaterials, 10(3), 420.
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2

Multiplex Immunofluorescence Profiling of Tissue Samples

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Tissue samples were fixed in formalin, embedded in paraffin, and sectioned. These specimens were incubated with the following antibodies: anti-CD3 (A045229–2; DAKO), anti-CD4 (ab133616; Abcam), anti-Pan-CK (ab27988; Abcam), anti-CD31 (3528; Cell Signaling Technology), anti-SLAMF7 (HPA055945; Sigma-Aldrich), anti-CX3CR1 (ab8021; Abcam), anti-T-bet (ab150440; Abcam), anti-GATA3 (MA1028; Invitrogen), anti-Ror gamma (ab212496; Abcam), anti-CXCR5 (clone: MAB190; R&D Systems), anti-FoxP3 (clone: 98377; Cell Signaling Technology), anti-CD8 (ab85792; Abcam), anti-PD1 (B13300; Lifespan Bioscience), anti-GZMB (ab4095; Abcam), anti-HLA-DR (ab20181; Abcam), anti-TGF-β (ab27969; Abcam) and anti-cleaved caspase-3 (9664; Cell Signaling Technology) followed by incubation with a secondary antibody using an OpalTM Multiplex Kit (Perkin Elmer). The samples were mounted with ProLongTM Diamond Antifade mountant containing DAPI (Invitrogen).
Kaneko N., Boucau J., Kuo H.H., Perugino C., Mahajan V.S., Farmer J.R., Liu H., Diefenbach T.J., Piechocka-Trocha A., Lefteri K., Waring M.T., Premo K.R., Walker B.D., Li J.Z., Gaiha G., Yu X.G., Lichterfeld M., Padera RF J.r, & Pillai S. (2022). Temporal changes in T cell subsets and expansion of cytotoxic CD4+ T cells in the lungs in severe COVID-19. Clinical Immunology (Orlando, Fla.), 237, 108991.
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3

Comprehensive Immunostaining Protocol

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Immunofluorescence staining analysis was performed on 40 μm free-floating sections. Primary antibodies, including chicken anti-GFP (Aves Labs, GFP-1020), goat anti-CD206 (R&D Systems, AF2535), rabbit anti-Cleaved Caspase-3 (Cell Signaling, 9661), mouse anti-Caspase-3 (Novus Biologicals, 31A1067), mouse anti-NeuN (Millipore, MAB377), rabbit anti-Tmem119 (Abcam, ab209064), mouse anti-LPL (Abcam, ab21356), rabbit anti-CX3CR1(Abcam, ab8021), rabbit anti-GFAP (Abcam, ab7260), mouse anti-iNOS (inducible nitric oxide synthase) (Abcam, ab49999), and goat anti-Iba1 (Abcam, ab5076), were used. Corresponding secondary antibodies, including 488- and 594-conjugated secondary antibodies, were purchased from Thermo Fisher Scientific, Alexa Fluor conjugates.
All images were processed with Image J for quantification analysis. The means were calculated from 3 randomly selected microscopic fields in the ipsilateral and contralateral cortex of each section, respectively, and 3 consecutive sections were analyzed for each brain. Data are expressed as mean numbers of cells per square millimeter.
Ye S.Y., Apple J.E., Ren X., Tang F.L., Yao L.L., Wang Y.G., Mei L., Zhou Y.G, & Xiong W.C. (2019). Microglial VPS35 deficiency regulates microglial polarization and decreases ischemic stroke-induced damage in the cortex. Journal of Neuroinflammation, 16, 235.
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4

Protein Expression Analysis via Western Blotting

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For the Western blot analyses, RIPA buffer containing protease inhibitors and phosphatase inhibitors (Roche) was used to prepare whole‐cell lysates. Twenty micrograms protein of lysates was separated by 10% SDS‐PAGE gels. After SDS‐PAGE electrophoresis, the polyvinylidene difluoride (PVDF) membrane (0.45 μm, Millipore) was put in 5% BSA for blocking at 37°C for 2 hours. After interacting with primary antibodies (CX3CR1 1:1000, ab8021, Abcam; MMP‐3 1:1000, YT4465, ImmunoWay; p‐c‐Src 1:1000, YP0077, ImmunoWay; c‐Src 1:1000, YT1139, ImmunoWay; MMP‐9 1:1000, ab76003, Abcam; p‐c‐abl 1:1000, YP0004, ImmunoWay; c‐abl 1:1000, YT0585, ImmunoWay; cortactin 1:500, sc‐55579, santa cruz; p‐cortactin 421 1:1000, YP0072, ImmunoWay) overnight at 4°C, 0.1% TBST was used to wash all membranes for 3 times, 5 minutes each time. IgG antibody of HRP‐antimouse or rabbit (1:3000, ZSGB‐BIO, Beijing, China) was used to combine primary antibodies at 37°C for 1 hour; the membranes were detected by chemiluminescence.
Fan M., Wu J., Li X., Jiang Y., Wang X., Bie M., Weng Y., Chen S., Chen B., An L., Zhang M., Huang G., Zhu M, & Shi Q. (2020). CX3CL1 promotes tumour cell by inducing tyrosine phosphorylation of cortactin in lung cancer. Journal of Cellular and Molecular Medicine, 25(1), 132-146.
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5

RSV-induced CX3CR1 Expression Analysis

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Following RSV infection, cells were recovered by trypsinization (0.25%), blocked with BSA, and incubated with anti-CX3CR1 antibody (Abcam ab8021) for 1 h at RT. Cells were analyzed for GFP and CX3CR1 on a BD LSRII. Data were analyzed using the FlowJo Software.
Anderson C.S., Chu C.Y., Wang Q., Mereness J.A., Ren Y., Donlon K., Bhattacharya S., Misra R.S., Walsh E.E., Pryhuber G.S, & Mariani T.J. (2019). CX3CR1 as a respiratory syncytial virus receptor in pediatric human lung. Pediatric research, 87(5), 862-867.
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6

Immunofluorescent and Immunoblot Analyses of GBM Tumor Markers

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Immunoblotting and immunofluorescent staining were performed as described65 (link), 66 (link). Specific antibodies against POSTN (Abcam, ab14041, 1: 400), GSC marker SOX2 (Millipore, AB5603, 1:200; Santa Cruz, sc-17320, 1:200) or OLIG2 (R&D systems, AF2418, 1:100), endothelial cell marker Glut1 (Millipore, 07-1401, 1:500) or CD31 (Dako, M082301, 1:100), and macrophage markers Iba1 (Abcam, ab5076, 1:200), CD11b (Abd serotec, MCA711GT, 1:100), Fizz1 (Abcam, ab39626, 1:100), CD163 (Santa Cruz, sc-33560, 1:100), F4/80 (Abd serotec, MCA497RT, 1:100), CX3CR1 (Abcam, ab8021, 1:100) or CCR2 (Abcam, ab32144, 1:100) were used for immunofluorescent staining on GBM tumor sections as indicated. Specific antibodies against POSTN (Abcam, ab14041, 1: 10,000), tubulin (Sigma, T8203, 1:10,000), phosphor-S473 Akt (Cell Signaling Technology, 9271, 1:1,000), Akt (Cell Signaling Technology, 9272, 1:1,000) were used for immunoblot analyses. To determine TAM density, cryosections of GBM tumors were co-stained with Iba1 (or CD11b) and Glut1. The numbers of Iba1+ (or CD11b+) cells were calculated by ImageJ. Glut1 positive areas were regarded as vessels and determined by ImageJ. For TAMs density in each sample, the number of Iba1+ (or CD11+) cells was divided by vessel area for normalization to exclude the individual variation in vascularization. The final outcome was defined as the TAM density.
Zhou W., Ke S.Q., Huang Z., Flavahan W., Fang X., Paul J., Wu L., Sloan A.E., McLendon R.E., Li X., Rich J.N, & Bao S. (2015). Periostin Secreted by Glioblastoma Stem Cells Recruits M2 Tumor-associated Macrophages and Promotes Malignant Growth. Nature cell biology, 17(2), 170-182.
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7

Quantifying Cellular Protein Expression

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Total cellular protein on the glass slides was extracted using RIPA lysis buffer containing protease and phosphorylase inhibitors (Thermo Fisher Scientific, USA). Protein concentration was measured using the Pierce BCA Protein Assay Kit according to the manufacturer’s instructions. Proteins were separated using 12% SDS-PAGE and transferred to polyvinylidene fluoride membranes (LOT 18071300, Roche) by electroblotting. Membranes were incubated in 5% skimmed milk in Tris buffered saline with Tween 20 (TBST) (Boshide, Wuhan, China) for 1 h to block nonspecific binding. Subsequently, the membranes were incubated overnight with either rabbit anti-CX3CR1 (1:1,000; ab8021, Abcam), NF-κB P65 (1:1,000), VCAM-1 (1:2,000), or rabbit anti-GAPDH (1:1,000; Goodhere, China) primary antibodies at 4 °C. The membranes were washed three times with TBST and incubated with secondary antibody conjugated to HRP (1:500; ZSGB-BIO, China) for 2 h. Protein bands were visualized using a color development solution (1:1) for 3 min. Images were acquired using a chemiluminescence imager (Bio-Rad).
Zhao Y., Ren P., Li Q., Umar S.A., Yang T., Dong Y., Yu F, & Nie Y. (2020). Low Shear Stress Upregulates CX3CR1 Expression by Inducing VCAM-1 via the NF-κB Pathway in Vascular Endothelial Cells. Cell Biochemistry and Biophysics, 78(3), 383-389.
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8

Immunohistochemistry for GBM Biomarkers

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IHC staining was carried out with the ABC kit and DAB kit (Vector Laboratories) according to manufacturer’s instructions. Primary antibodies for POSTN (ab14041, 1:400), Iba1 (ab5076, 1:400), CX3CR1 (ab8021, 1:100), CCR2 (ab32144, 1:100) and Ki67 (ab15580, 1:400) from Abcam were used for the IHC staining. To study the correlation between POSTN level and TAM recruitment, the sequential sections of primary GBM microarrays were stained with antibodies against POSTN or Iba1, respectively. The IHC staining of POSTN was evaluated by two independent pathologists. The samples with a score of 0~1 were regarded as low expression and those with a score of 2~4 were regarded as high expression. For Iba1 evaluation, 3 random areas from a single section were checked for Iba1+ cells. If the average number of Iba1+ cells was less than 100 Iba1+ cells in 4.2mm2 (2.36×1.78 mm2), the sample was ascribed to Iba1 low. Otherwise the sample was classified as Iba1 high.
Zhou W., Ke S.Q., Huang Z., Flavahan W., Fang X., Paul J., Wu L., Sloan A.E., McLendon R.E., Li X., Rich J.N, & Bao S. (2015). Periostin Secreted by Glioblastoma Stem Cells Recruits M2 Tumor-associated Macrophages and Promotes Malignant Growth. Nature cell biology, 17(2), 170-182.
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9

Immunohistochemistry for GBM Biomarkers

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IHC staining was carried out with the ABC kit and DAB kit (Vector Laboratories) according to manufacturer’s instructions. Primary antibodies for POSTN (ab14041, 1:400), Iba1 (ab5076, 1:400), CX3CR1 (ab8021, 1:100), CCR2 (ab32144, 1:100) and Ki67 (ab15580, 1:400) from Abcam were used for the IHC staining. To study the correlation between POSTN level and TAM recruitment, the sequential sections of primary GBM microarrays were stained with antibodies against POSTN or Iba1, respectively. The IHC staining of POSTN was evaluated by two independent pathologists. The samples with a score of 0~1 were regarded as low expression and those with a score of 2~4 were regarded as high expression. For Iba1 evaluation, 3 random areas from a single section were checked for Iba1+ cells. If the average number of Iba1+ cells was less than 100 Iba1+ cells in 4.2mm2 (2.36×1.78 mm2), the sample was ascribed to Iba1 low. Otherwise the sample was classified as Iba1 high.
Zhou W., Ke S.Q., Huang Z., Flavahan W., Fang X., Paul J., Wu L., Sloan A.E., McLendon R.E., Li X., Rich J.N, & Bao S. (2015). Periostin Secreted by Glioblastoma Stem Cells Recruits M2 Tumor-associated Macrophages and Promotes Malignant Growth. Nature cell biology, 17(2), 170-182.
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10

CTC Enrichment and CX3CR1 Detection

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Breast cancer patients were enrolled in a prospective biomarker study. All subjects gave informed consent; the study was approved by the Institutional Review Board at Feinberg School of Medicine. The clinical trial enrolled patients with stage III and IV disease irrespective of subtype, about to start next systemic therapy. Blood form a breast cancer patient diagnosed with ER+/PR+/HER2− Intraductal Carcinoma (IDC), staging T4N1M1, was first processed for enrichment using the Parsortix™ system (Angle plc, Surrey, UK) and then CTCs were collected using the Captor® system (Clearbridge Biomedics, Singapore). Detection of CX3CR1 was achieved by on-chip immunofluorescence staining using a primary antibody (ab8021, 1ng/ml) from Abcam (Cambridge, MA) followed by Tyramide Signal Amplification with AlexaFluor™ 594 (B40944, ThermoFisher Scientific, Waltham, MA). For fluorescence microscopy and imaging we used an EVOS FL system (ThermoFisher Scientific, Waltham, MA).
Qian C., Worrede-Mahdi A., Shen F., DiNatale A., Kaur R., Zhang Q., Cristofanilli M., Meucci O, & Fatatis A. (2018). Impeding Circulating Tumor Cell Reseeding Decelerates Metastatic Progression and Potentiates Chemotherapy. Molecular cancer research : MCR, 16(12), 1844-1854.
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