Alexa fluor 488 anti mouse f4 80

Manufactured by BioLegend

Sourced in United States

Alexa Fluor 488 anti-mouse F4/80 is a fluorescently labeled antibody that binds to the F4/80 antigen, a glycoprotein expressed on the surface of murine macrophages.

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5 protocols using alexa fluor 488 anti mouse f4 80

Pancreatic Immune Cell Profiling

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Freshly harvested pancreatic tissue samples were digested in 0.75 mg/mL collagenase-P (Roche Basel, Switzerland) solution at 37°C for 15 min. Subsequently, tissue was homogenized in gentleMACS™ Dissociators (Miltenyi Biotec, Bergisch Gladbach, Germany) and filtered through 75 μm filter screen with phosphate buffer saline (PBS). Single-cell suspensions were incubated for 15 min at room temperature in PBS with the following antibodies: PE/Cy7 anti-mouse CD45, Alexa Fluor 488 anti-mouse F4/80, Brilliant Violet 421 anti-mouse/human CD11b, PE anti-mouse CD206 (MMR), Alexa Fluor 647 anti-mouse Ly6G and APC anti-mouse CD86 from Biolegend (CA, USA). Isotype-matched controls were included in all experiments. Gating methods of fluorescence-activated cell sorting were programmed as CD45⁺CD11b⁺Ly6G⁺ (for neutrophils), CD45⁺CD11b⁺F4/80⁺CD86⁺ (for M1 macrophages) and CD45⁺CD11b⁺F4/80⁺CD206⁺ (for M2 macrophages). Stained cells were analyzed on an Attune NxT flow cytometer (Thermo Fisher Scientific, MA, USA).

Ren Z., Li H., Zhang M., Zhao Y., Fang X., Li X., Chen W., Zhang H., Wang Y., Pan L.L, & Sun J. (2018). A Novel Derivative of the Natural Product Danshensu Suppresses Inflammatory Responses to Alleviate Caerulein-Induced Acute Pancreatitis. Frontiers in Immunology, 9, 2513.

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Comprehensive Inflammatory Cell Profiling

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All stains were performed on formalin-fixed paraffin-embedded skin sections. Inflammatory infiltrates were initially assessed by H&E staining. For cell-specific stains, sections were deparaffinized and slides boiled in sodium citrate buffer as described previously. For CD3 staining, rabbit anti-CD3 antibody (Abcam, Cambridge, United Kingdom, ab5690, 1:150) and an EnVisionþ System-HRP (DAB) kit (Dako, Carpinteria, CA, K4011) were used according to the manufacturer's instructions. For staining of other inflammatory cell markers, sections were treated with MOM blocking buffer (Vector Laboratories, Burlingame, CA, PK2200) at room temperature for 1 hour before incubation separately with primary antibodies diluted in blocking buffer (Alexa Fluor 488 anti-mouse Ly-6G, 1:400, Biolegend, San Diego, CA, 108419; Alexa Fluor 488 anti-mouse F4/80, 1:600, Biolegend 123119; Alexa Fluor 594 anti-mouse CD272 (BTLA), 1:200, Biolegend 139111) overnight at 4 C. After washing with PBS containing 0.05% Tween-20, ProLong Gold Antifade Mountant with DAPI was applied and slides were stored in the dark at 4 C.
Methods for staining of mast cells and Trichrome staining are provided in Supplementary Materials and Methods.

Rahman H., Kumar D., Liu T., Okwundu N., Lum D., Florell S.R., Burd C.E., Boucher K.M., VanBrocklin M.W, & Grossman D. (2021). Aspirin Protects Melanocytes and Keratinocytes against UVB-Induced DNA Damage In Vivo. The Journal of investigative dermatology, 141(1).

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Isolation and Phenotyping of Tumor-Associated Macrophages

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The tumors were removed, cut into small pieces with a scalpel, added to HBSS culture containing 1 mg/mL collagenase (Sigma) and 0.1 mg/mL DNAase (Sigma), and incubated for 1 h at 37°C for digestion. The digests were sieved with 70 μM filters and the cells were collected by centrifugation and washed with PBS containing 2% FBS to prepare a tumor single‐cell suspension. Cell suspensions were then incubated with FcRblock (1 μg/test; BioLegend, #101319) for 10 min at 4°C, followed by treatment with CD45 (PE anti‐mouse CD45, 0.5 μg/test; BioLegend, #157603), F4/80 (Alexa Fluor® 488 anti‐mouse F4/80; 1 μg/test, BioLegend, #123119), CD11b (PerCP‐Cy5.5 anti‐mouse CD11b; 0.5 μg/test, BioLegend, #101227) antibodies at 4°C for 30 min staining. Then the cells were washed with PBS, fixed with Fixation Buffer (Fixation Buffer, BioLegend, #420801), and cell penetration was performed using 0.5% Triton X‐100. After cell resuspension with buffer containing 2% FBS in PBS, APC anti‐mouse CD206 antibody (0.5 μg/tests, BioLegend, #141707) was added and incubated for 30 min at room temperature and protected from light. Finally, the treated cell suspension was placed into a flow cytometer for detection.

Zhou H., Su D., Chen Y., Zhang Y, & Huang P. (2023). KCND2: A prognostic biomarker and regulator of immune function in gastric cancer. Cancer Medicine, 12(15), 16279-16294.

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Immunofluorescence Staining of Mouse Ear Cryosections

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Explanted mice ears where cut by a biopsy pouch centered in the middle zone (diameter 8 mm), washed twice with PBS, embedded at the hedge in O.C.T. (Tissue-Tek O.C.T. Compound, Sakura Finetek), and instantly frozen in liquid nitrogen. 8-μm thick slides were obtained cutting ears block with a cryostat at −20 °C. For H&E staining, slides were thawed, hydrated, washed and stained with hematoxylin and eosin (Sigma-Aldrich). Immunofluorescence staining has been performed on consecutive ear sections. Briefly, slides were thawed and blocked with goat serum 5% (Sigma-Aldrich) PBS-T 1 × solution. After washing, they were incubated overnight at 4° with anti-macrophage antibody and anti- CD206 (Alexa Fluor 488 anti-mouse F4/80 and Brilliant Violet 605 anti-mouse CD206 Biolegend). Excess of the anti-neutrophil antibody was washed out with PBS 1X. Cells nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI). Slides were sealed with ProLong Gold antifade reagent (Life Technologies). Images were captured with a Nikon Eclipse Ti Inverted Fluorescent Microscope.

Corradetti B., Taraballi F., Martinez J.O., Minardi S., Basu N., Bauza G., Evangelopoulos M., Powell S., Corbo C, & Tasciotti E. (2017). Hyaluronic acid coatings as a simple and efficient approach to improve MSC homing toward the site of inflammation. Scientific Reports, 7, 7991.

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Characterizing Leptin-Responsive Immune Cells

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LepR expression on naïve CD4⁺ T cells and T_FH cells from WT mice were stained with leptin receptor (LepR) (Polyclone, R&D) with secondary antibody Alexa Fluor 488 Donkey Anti-Goat IgG H&L(Abcam).
Leptin-secreting cells in the spleen of WT mice were stained with the following antibodies conjugated with fluorochromes: Alexa Fluor 488 anti-mouse IgM (Clone: RMM-1, Biolegend), Brilliant Violet 421 anti-mouse CD4 (Clone: GK1.5, Biolegend), Alexa Fluor 555 leptin (Polyclone, Bioss), Brilliant Violet 421 anti-mouse CD19 (Clone: 6D5, Biolegend), Alexa Fluor 488 anti-mouse F4/80 (Clone: BM8, Biolegend), Nuclei were identified by DAPI staining. Images were obtained with confocal microscopy (Zeiss LSM 710, Zeiss), and analysed with Photoshop CS4 software (Adobe) and Zen software (Zeiss).

Deng J., Chen Q., Chen Z., Liang K., Gao X., Wang X., Makota F.V., Ong H.S., Wan Y., Luo K., Gong D., Yu X., Camuglia S., Zeng Q., Zhou T., Xue F., He J., Wei Y., Xiao F., Ma J., Hill D.L., Pierson W., Nguyen T.H., Zhou H., Wang Y., Shen W., Sun L., Li Z., Xia Q., Qian K., Ye L., Rockman S., Linterman M.A., Kedzierska K., Shen N., Lu L, & Yu D. (2021). The metabolic hormone leptin promotes the function of TFH cells and supports vaccine responses. Nature Communications, 12, 3073.

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