Paraffin or cryostat sections were prepared as described previously (35 (link)). Paraffin sections were stained with hematoxylin and eosin (H&E). For fluorescence double staining, cryostat sections were incubated with anti-iNOS antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and anti-F4/80 antibodies (BioLegend, San Diego, CA, USA), followed by incubation with Alexa Fluor 488- or 594-conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA, USA). Sections were evaluated under a microscope (DP71, OLYMPUS) of both bright-field and fluorescence microscopy (200 × magnification).
Anti f4 80 antibody
Manufactured by BioLegend
Sourced in United States
The Anti-F4/80 antibody is a laboratory reagent used to detect and identify the F4/80 antigen, which is expressed on the surface of mature mouse macrophages. This antibody can be used in various immunological techniques, such as flow cytometry, immunohistochemistry, and western blotting, to study the presence and distribution of macrophages in biological samples.
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Lab products found in correlation
Anti cd206 antibody, by BioLegend (3 mentions)
Fitc conjugated annexin 5 antibody, by BioLegend (2 mentions)
Tunel reagent, by Roche (2 mentions)
Anti mac2 antibody, by Cedarlane (2 mentions)
Dexamethasone (dex), by Merck Group (2 mentions)
Countess 2, by Thermo Fisher Scientific (2 mentions)
Anti erk1 2, by Cell Signaling Technology (2 mentions)
Anti dusp4, by Abcam (2 mentions)
Anti phospho erk1 2, by Cell Signaling Technology (2 mentions)
Anti tgf β1, by Abcam (2 mentions)
Alexa fluor 488 or 594 conjugated secondary antibodies, by Jackson ImmunoResearch (1 mentions)
Anti inos antibody, by Santa Cruz Biotechnology (1 mentions)
Flow cytometry, by Agilent Technologies (1 mentions)
Fetal bovine serum (fbs), by Lonza (1 mentions)
Dulbecco modified eagle medium (dmem), by Lonza (1 mentions)
Penicillin streptomycin mixture, by Lonza (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Cfx connect real time pcr detection system, by Bio-Rad (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Itaq sybr green supermix, by Bio-Rad (1 mentions)
14 protocols using anti f4 80 antibody
1
Histological Analysis of Tissue Sections
2
Macrophage Polarization in Inflammation
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Initially, LPS was used as the trigger for the status of inflammation. In a 6-well plate, RAW 264.7 cells were seeded at a density of 5 × 10 5 cells/well and cultivated for a day. Subsequently, LPS (1 µg/mL) was added to activate the cells for 12 h to mimic the inflammation in vitro before various NPs were added. Following a 24hour incubation period, the cells were separated by centrifugation, subjected to two rounds of cold PBS washing, and stained using anti-iNOS antibodies (Biolegend, USA) labeled with APC, anti-F4/80 antibodies (Biolegend, USA) labeled with FITC, and anti-CD206 antibodies (Biolegend, USA) labeled with PE. Utilizing flow cytometry (Agilent, USA), the proportion of polarized macrophages was determined. Using qPCR, the mRNA expression levels of IL-1β, IL-6, TNF-α, IL-10, TGF-β, and Arg-1 were used to assess the M1/M2 phenotypic inflammatory response of RAW 264.7.
3
Culturing Primary Bone Marrow-Derived Macrophages
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The macrophage colony-stimulating factor (M-CSF)-producing L929 fibroblast cell line (a kind gift from Csaba Vizler, Biological Research Centre, Szeged) was used to obtain L929-conditioned medium. To this end, confluent cultures were incubated with nonsupplemented Dulbecco’s modified Eagle’s medium (DMEM; Lonza) in 75-ml tissue culture flasks for 10 days. Cell culture supernatants were subsequently sterile filtered, aliquoted, and kept at −20°C until utilization.
Based on previous studies (25 (link), 80 ), primary bone marrow-derived macrophages (BMDMs) were cultured from the bone marrows of 8- to 15-week-old female and male bone marrow chimeric mice in BMDM medium (80% [vol/vol] DMEM supplemented with 10% heat-inactivated fetal bovine serum [FBS; Lonza] and 1% penicillin-streptomycin mixture [Lonza]; 20% [vol/vol] L929-conditioned medium) for 7 to 9 days in 96-, 24-, or 12-well plates, according to the experiment. Fresh medium was added every other day.
We checked the functionality of the macrophage culturing method by immunostaining with anti-CD11b (Sony) and anti-F4/80 antibodies (BioLegend) or isotype controls (Sony and BioLegend) followed by flow cytometric analysis where CD11b+ F4/80+ double-positive cells were considered macrophages (81 (link)). As the proportion of these cells was over 80% in the case of all genotypes (data not shown), we regarded the method as functional.
Based on previous studies (25 (link), 80 ), primary bone marrow-derived macrophages (BMDMs) were cultured from the bone marrows of 8- to 15-week-old female and male bone marrow chimeric mice in BMDM medium (80% [vol/vol] DMEM supplemented with 10% heat-inactivated fetal bovine serum [FBS; Lonza] and 1% penicillin-streptomycin mixture [Lonza]; 20% [vol/vol] L929-conditioned medium) for 7 to 9 days in 96-, 24-, or 12-well plates, according to the experiment. Fresh medium was added every other day.
We checked the functionality of the macrophage culturing method by immunostaining with anti-CD11b (Sony) and anti-F4/80 antibodies (BioLegend) or isotype controls (Sony and BioLegend) followed by flow cytometric analysis where CD11b+ F4/80+ double-positive cells were considered macrophages (81 (link)). As the proportion of these cells was over 80% in the case of all genotypes (data not shown), we regarded the method as functional.
4
Isolation and Analysis of Tumor-Associated Macrophages
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Dissociated cells from primary tumors were incubated with anti-F4/80 antibodies (Biolegend; clone BM8; 123102) bound to sheep anti-rat IgG-conjugated beads (Life Technologies; 11035) and isolated according to manufacturer’s instructions. RNA was isolated from macrophages and tumor cells using TRIzol (Life Technologies; 15596026) and purified using Qiagen RNeasy Mini Kit (Qiagen; 74104). RNA was reverse transcribed using the High Capacity cDNA Reverse Transcription Kit (Applied Biosciences; 4368814) and Techne Thermal Cycler or Biometra Tone Thermal Cycle. Quantitative PCR was performed using iTaq SYBR Green Supermix (Bio-Rad; 172-5121) with a Bio-Rad CFX Connect Real-Time PCR Detection System (Bio-Rad). Transcripts were normalized to housekeeping genes cyclophilin or ribosomal protein lateral stalk subunit P0 (Rplp0), and data was analyzed using the ∆∆Cq method. Primer sequences are listed in Table S1 .
5
Immunohistochemical Analysis of IL-33 in Liver
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Liver samples were fixed in 4% paraformaldehyde and then paraffin-embedded using a standard method. Fixed tissue samples were sectioned and stained with hematoxylin and eosin (H&E). Pictures were taken using an Olympus BX-53 microscope. For immunohistochemical labeling, after preincubation with 3% hydrogen peroxide and blocking with 2% bovine serum albumin (Amresco, Solon, OH, USA), sections were stained with anti-IL-33 (WC3234729; Invitrogen Life Technologies, Carlsbad, CA, USA), anti-CES1 (ERP1375Y; Abcam, Cambridge, UK), or anti-F4/80 antibody (B281627; Biolegend, San Diego, CA, USA) overnight at 4 °C. After washing, sections were incubated with respective secondary antibodies. For immunofluorescent labeling, paraffin-embedded human liver tissue sections were deparaffinized, rehydrated, and antigen was retrieved by Tris- ethylenediaminetetraacetic acid buffer for 95°C for 30 m. Then, the sections were incubated with anti-IL-33 antibody (WC3234729; Invitrogen) at 4°C overnight, secondary antibody the next day, and the mounted with 4′,6-diamidino-2-phenylindole. Positive staining was visualized using an Olympus BX-51 fluorescence microscope. The total stained area was analyzed by ImageJ (NIH, Bethesda, MD, USA).
6
Isolation of Primary Kupffer Cells
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Primary Kupffer cells were isolated separately from C57BL/6 mice in the Con group, LPS group, DHA + LPS group, and AA + LPS group as previously described42 (link). Briefly, the liver was perfused with 10 mL of phosphate-buffered saline and then digested with 0.1% type IV collagenase. Following digestion, the liver homogenate was filtered through a 75-μm stainless steel wire mesh to remove undigested tissue. The cell suspension was centrifuged at 50g (Eppendorf 5810 R, Germany) for 5 min at 4 °C. The top suspension was separated with 60% Percoll and then centrifuged at 2500g for 25 min. The darker layer in the middle comprised Kupffer cells. Kupffer cells were identified by flow cytometry using a monoclonal anti-F4/80 antibody (123114; Biolegend, California, USA) conjugated with PE/Cyanine7 (PE-Cy7). Flow cytometry was performed for detecting the percentage of F4/80-positive cells. Briefly, the collected cells were incubated with the antibody (1:100 dilution) for 30 min at 37 °C. The data were collected using a flow cytometer.
7
Dexamethasone-Induced Thymus Apoptosis in Mice
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8–10 weeks old male mice were injected i.p. with 250 uL PBS containing 250 mg dexamethasone (Sigma-Aldrich) dissolved in DMSO. Eighteen hours after injection, the mice were euthanized, and thymi were harvested and weighed. One lobe of the thymus was mechanically disaggregated, and cells number was determined using Countess II (Invitrogen). 1×106 cells were stained with either FITC-conjugated Annexin V antibody (Biolegend) or anti-F4/80 antibody (Biolegend) to determine apoptotic cells and macrophage number, respectively. The other thymus lobe was formalin-fixed, paraffin-embedded, and sectioned, followed by immunostaining the sections with TUNEL reagent (Roche), anti-TGFβ1 (Abcam), anti-phospho-ERK1/2 (Cell Signaling), anti-ERK1/2 (Cell Signaling), anti-DUSP4 (Abcam), and/or anti-Mac2 antibodies (Cedarlane). In situ efferocytosis was quantified by counting TUNEL+ cells that were associated with Mac2+ cells versus macrophage-free TUNEL+ cells. Macrophage-associated apoptotic cells followed the criteria of TUNEL+ nuclei surrounded by or in contact with neighboring Mac2+ macrophages. Free apoptotic cells exhibited nuclear condensation and were not in contact with neighboring macrophages.
8
Dexamethasone-Induced Thymus Apoptosis in Mice
9
Liver Histopathological Analysis
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Liver samples were fixed overnight in 4% paraformaldehyde for cryosections, or 10% neutral buffered formalin for paraffin embedding. Endogenous biotin-blocked, 4-µm-thick paraffin sections were stained with haematoxylin, anti-C3d antibody (R&D Systems, Inc.) or anti-F4/80 antibody (Biolegend, Inc.)37 (link),38 (link). Six-µm-thick cryosections were stained with Oil Red O (Sigma-Aldrich) and Mayer's Hematoxylin Solution (Fujifilm Wako Pure Chemical Corporation)39 (link). Captured images were analysed using the BZ-X800 Analyzer (Keyence Corporation) or ImageJ (Fiji)40 (link). C3d-positive area and F4/80-positive cells per hepatocyte were calculated.
10
Atherosclerotic Lesion Analysis: Comprehensive Protocols
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