O.C.T-embedded tumor sections (7 μm-thick) were incubated with blocking solution (PBS, 5% goat serum (G9023, Sigma-Aldrich), 5% rat serum (R9759, Sigma-Aldrich), 1% FCS) for 1 hour at room temperature before incubation with primary antibody overnight at 4°C [anti–tenascin-C from Sigma-Aldrich (MTn-12)) at 10 μg/mL ; anti–F4/80-PE (BM8) and anti–CD206-APC (C068C2) from Biolegend] both at 1/100. The slides were then incubated with secondary antibody for 1 hour at room temperature (anti-IgG1-AF488, Biolegend) at 1/200, counterstained with DAPI (Thermofisher), and embedded with Fluoromount-G™ (Invitrogen). The fluorescent signal was analyzed with a Zeiss Axio Imager microscope. Five random 20x-fields per section were analyzed by ImageJ (National Institutes of Health, USA) to assess the infiltration of macrophages, as well as their spatial distribution between the tumor nest and the stroma (1 section per tumor, 5 tumors per group). Macrophage subsets and tenascin-C localization in the tumor were also analyzed and displayed as a linescan using FiJi software.
Anti f4 80 pe bm8
Manufactured by BioLegend
Anti-F4/80-PE (BM8) is a flow cytometry reagent used to detect the F4/80 antigen, a cell surface protein expressed on mouse macrophages. It is conjugated with the fluorescent dye phycoerythrin (PE) for direct detection by flow cytometry.
Automatically generated - may contain errors
Lab products found in correlation
R9759, by Merck Group (1 mentions)
Mtn 12, by Merck Group (1 mentions)
Anti cd206 apc c068c2, by BioLegend (1 mentions)
Axioimager microscope, by Zeiss (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Fluoromount g, by Thermo Fisher Scientific (1 mentions)
G9023, by Merck Group (1 mentions)
Anti b220 pe ra3 6b2, by BioLegend (1 mentions)
Anti b220 fitc ra3 6b2, by BD (1 mentions)
Anti cd25 pe clone pc61, by BioLegend (1 mentions)
Anti cd11b bv421 clone m1 70, by BioLegend (1 mentions)
Anti cd11b fitc clone m1 70, by BioLegend (1 mentions)
Anti cd27 apc clone l g 3a10, by BioLegend (1 mentions)
Anti cd8 fitc clone 53.6.7, by BioLegend (1 mentions)
Facscanto 2, by BD (1 mentions)
Apc cy7 anti cd45 30 f11, by BioLegend (1 mentions)
Fortessa, by BD (1 mentions)
Anti cd3 bv421 clone 17a2, by BioLegend (1 mentions)
3 protocols using anti f4 80 pe bm8
1
Immunofluorescent Profiling of Tumor Macrophages
2
Comprehensive Immune Cell Analysis
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The spleen was isolated and mashed into RPMI 1640 medium containing 2% FBS. After erythrocytes were lysed, a single cell suspension of splenocytes was prepared and then stained with the cell surface markers. For GC B cell staining, cells were stained with anti-B220-PE (RA3-6B2, Biolegend), anti-Fas-APC (15A7, eBioscience), and anti-GL-7-FITC (GL-7, Biolegend). For marginal zone B (MZB) cell staining, cells were stained with anti-B220-FITC (RA3-6B2, BD), anti-CD21-APC (7G6, BD), and anti-CD23-PE (B3B4, BD). For IgG-secreting plasma cells, after surface staining of anti-CD138-APC (IB17-R0268, Miltenyi), cells were fixed, permeabilized, and stained for anti-IgG-FITC (Poly4060, BD). For MZM staining, cells were stained with anti-F4/80-PE (BM8, Biolegend), anti-CD11b-Alexa Fluor 488 (M1/70, Biolegend), anti-SIGNR1-APC (22D1, eBioscience), and anti-MHC II (I-Ab)-eFluor 450 (AF6-120.1, eBioscience). All samples were detected on a flow cytometer (Beckman Coulter, USA) and analyzed with Cytexpert 2.0 software.
3
Multiparameter Flow Cytometry Analysis
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For FACS analysis, the following antibodies were used: anti-CD3-Bv421-(clone 17A2, Biolegend), anti-CD4-Bv421 (clone RM 4-5, eBioscience), anti-CD8-FITC (clone 53-6.7, Biolegend), anti-CD25 PE (clone PC61, Biolegend), anti-CD27-APC (clone LG.3A10, Biolegend), anti-CD11b-FITC (clone M1/70, Biolegend), anti-CD11b-Bv421 (clone M1/70, Biolegend), anti-FceRIα-PE (MAR-1, Biolegend), anti-F4/80-PE (BM8, Biolegend), anti-CD45-APC/Cy7 (30-F11, Biolegend), anti-CD107a-FITC (1D4B, Biolegend) and anti-P2X7-AF647 (clone Hano44, UKE) (Adriouch et al., 2005) (link). Flow cytometric analyses were performed on a BD Fortessa (Beckton Dickinson) or a BD FACS CantoII (Beckton Dickinson).
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