Peritoneal macrophage and stromal vascular (SV) cells of epididymal adipose tissues were fractionated as described[18 (link),19 (link)]. Briefly, to get peritoneal macrophage, 5 ml of cold phosphate buffer saline (PBS) was injected into mouse peritoneal cavities immediately after anesthesia. After shaking the mice for 2-3 min, peritoneal fluid was harvested and spun down for peritoneal macrophages at 500 g for 5 min at 4 °C. The stromal vascular cells were isolated from the equal mass of epididymal adipose tissues using the collagenase digestion method. For flow cytometry analysis, same quantity cells (1 × 106) were subsequently re-suspended and stained with appropriate antibodies (F4/80 and CD11c for M1 type macrophage, or F4/80 and CD206 for M2 type macrophage) as described in our previous study[20 (link)]. Antibody information used in flow cytometry analysis is as follows: PE anti-mouse F4/80 antigen (eBioscience, San Diego, CA), FITC anti-mouse CD11c antigen (BD Bioscience, San Jose, CA), purified CD16/CD32 antigen (BD Bioscience, San Jose, CA), and APC anti-mouse CD206 antigen (BD Bioscience, San Jose, CA). All data were collected using FACScan and analyzed using CellQuest software (BD Biosciences, San Jose, CA).
Pe anti mouse f4 80 antigen
Manufactured by Thermo Fisher Scientific
Sourced in United States
The PE anti-mouse F4/80 antigen is a fluorescent-labeled antibody that specifically binds to the F4/80 antigen, a cell surface glycoprotein expressed on mature macrophages and microglia in mice. This antibody can be used in flow cytometry applications to identify and quantify mouse macrophage populations.
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Lab products found in correlation
Cellquest software, by BD (3 mentions)
Fitc anti mouse cd11c antigen, by BD (3 mentions)
Apc anti mouse cd206 antigen, by BD (3 mentions)
Purified cd16 cd32 antigen, by BD (2 mentions)
Facscan, by BD (1 mentions)
Nonspecific igg, by BD (1 mentions)
Anti ki67, by Leica (1 mentions)
Ncl ki67p, by Leica (1 mentions)
U rfl t microscope, by Olympus (1 mentions)
Anti pcna, by Cell Signaling Technology (1 mentions)
Anti akt, by Cell Signaling Technology (1 mentions)
Anti p akt, by Cell Signaling Technology (1 mentions)
Anti c ebpα, by Santa Cruz Biotechnology (1 mentions)
Ab26721, by Abcam (1 mentions)
Anti β tubulin, by Merck Group (1 mentions)
Anti β actin, by Santa Cruz Biotechnology (1 mentions)
Anti gdf3, by R&D Systems (1 mentions)
Anti pparγ, by Santa Cruz Biotechnology (1 mentions)
Anti brd4 a301 985a, by Fortis Life Sciences (1 mentions)
Anti atgl, by Santa Cruz Biotechnology (1 mentions)
6 protocols using pe anti mouse f4 80 antigen
1
Isolation and Characterization of Murine Peritoneal Macrophages and Adipose Stromal Cells
2
Isolation and Characterization of Mouse Peritoneal Macrophages
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PM were isolated as described previously [41 (link)]. Briefly, about 5 mL of cold phosphate buffer saline (PBS) was injected into mouse abdominal cavities. After vigorously shaking the mice for 2 min, solution in abdominal cavity (the PBS containing PM) was carefully collected. PM was obtained by centrifugation at 1000 g for 5 min. For flow cytometry analysis, equal amounts of the PM cells (1 × 106 in 100 μL PBS) were incubated with appropriate antibodies; PE anti-mouse F4/80 antigen (eBioscience, San Diego, CA, USA), FITC anti-mouse CD11c antigen (BD Bioscience, San Jose, CA, USA), and APC anti-mouse CD206 antigen (BD Bioscience). The flow cytometry data were collected using a FACScan and analyzed with Cell Quest software (BD Biosciences). Macrophages labeled with F4/80+CD11c+CD206− were counted as pro-inflammatory M1-like macrophages and those labeled with F4/80+CD11C−CD206+ were counted as anti-inflammatory M2-like macrophages.
3
Isolation and Characterization of Adipose SVF
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Stromal vascular fraction was isolated as described previously [65 (link), 66 (link)]. Briefly, 1g of epididymal adipose tissue was dissected and minced in Krebs-Ringer bicarbonate buffer (KRB) containing 1 mg/ml collagenase Type I (Worthington Chemicals, Lakewood, NJ). The solution was incubated in 37°C water bath for 30 minutes. The tissue slurry was then filtered through nylon mesh to remove undigested tissue, and centrifuged at 2200 rpm to fractionate adipocytes and stromal vascular fraction (SVF). SVF cells (1 × 106 in a volume of 100 μl of PBS) were incubated with antibodies for flow cytometry analysis. The antibodies included PE anti-mouse F4/80 antigen (eBioscience, San Diego, CA), FITC anti-mouse CD11c antigen (BD Bioscience, San Jose, CA), purified CD16/CD32 antigen (BD Bioscience, San Jose, CA), and APC anti-mouse CD206 antigen (BD Bioscience, San Jose, CA). Cells were incubated with nonspecific IgG to assess background fluorescence (BD Bioscience, San Jose, CA). The data were collected using a FACScan and analyzed using CellQuest software (BD Biosciences, San Jose, CA).
4
Analyzing Liver Pathology Using IHC and Microscopy
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HE and immunohistochemistry were performed on formalin-fixed paraffin-embedded liver sections as described previously [46 (link)]. The following antibodies were used for staining: anti-α-SMA (Abcam, ab124964), anti-PCNA (Cell Signaling Technology, 2586), and anti-Ki67 (Novocastra, NCL-Ki67p).
To detect collagen deposition, paraffin sections were stained with Sirius red dissolved in picric acid solution according to the manufacturer’s recommendations.
For F4/80 immunofluorescence staining, liver cryosections were fixed in precooled acetone for 10 minutes and washed with phosphate-buffered saline (PBS) three times. After immersion in diluted normal goat serum, sections were sequentially incubated with anti-mouse F4/80 antigen PE (eBioscience, 12–4801) overnight at 4°C. Nuclei were stained with DAPI for 5 minutes after two washes with PBS.
TUNEL assays were performed using an Apoptosis DNA Fragmentation Assay Kit (Clontech, #630107) to detect apoptotic cells. All images were visualized using a U-RFL-T microscope (Olympus, Tokyo, Japan). The positive cells or areas were counted or measured in at least five fields on each slide using ImageJ (NIH) software.
To detect collagen deposition, paraffin sections were stained with Sirius red dissolved in picric acid solution according to the manufacturer’s recommendations.
For F4/80 immunofluorescence staining, liver cryosections were fixed in precooled acetone for 10 minutes and washed with phosphate-buffered saline (PBS) three times. After immersion in diluted normal goat serum, sections were sequentially incubated with anti-mouse F4/80 antigen PE (eBioscience, 12–4801) overnight at 4°C. Nuclei were stained with DAPI for 5 minutes after two washes with PBS.
TUNEL assays were performed using an Apoptosis DNA Fragmentation Assay Kit (Clontech, #630107) to detect apoptotic cells. All images were visualized using a U-RFL-T microscope (Olympus, Tokyo, Japan). The positive cells or areas were counted or measured in at least five fields on each slide using ImageJ (NIH) software.
5
Antibody Validation for Immunoblotting and ChIP
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Antibodies for immunoblotting were as follows: anti-β-actin (sc-47778, Santa Cruz); anti-β-Tubulin (T8328, Sigma-Aldrich); anti-AKT (9272, Cell Signaling Technology); anti-p-AKT (4051, Cell Signaling Technology); anti-Gdf3 (AF958, R&D Systems); anti-ATGL (sc-8020, Santa Cruz); anti-C/EBPα (sc-365318, Santa Cruz); and anti-PPARγ (sc-7273, Santa Cruz). Antibodies for ChIP were as follows: anti-Brd4 (A301-985A, Bethyl Laboratories Inc); anti-PPARγ (ab41928, Abcam); and anti-RNA polymerase II (ab26721, Abcam). Antibodies for FACS flow cytometry were as follows: anti-mouse CD45.2 PerCP-Cyanine5.5 (45-0454, eBioscience); anti-mouse F4/80 antigen PE (12-4801, eBioscience); anti-mouse CD11b APC-eFluor 780 (47-0112, eBioscience); anti-mouse CD11c Brilliant Violet 60 (117334, BioLegend); and anti-Mouse CD301Alexa Fluor 647 (MCA2392A647, Bio-Rad).
6
GFP-HCT-116 Cell Line Characterization
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The GFP-HCT-116 cell line (Retroviral GFP transduction) was purchased from Nanjing Origin Biotechnology Co. Ltd. (Nanjing, Jiangsu, China). Antibodies for flow cytometry were as follows: Mouse FcR Blocking (Miltenyi, 130–092–575), CD45-PE-Vio770 mouse (Miltenyi, 130–105-462), Anti-Mouse F4/80 Antigen PE (eBioscience, 12–4801-80), Anti-Mouse CD11b APC (eBioscience, 17–0112-81), Anti-Mouse Ly-6G-FITC (eBioscience, 11–9668-82).
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