Immunostaining was performed on human breast tissue array (Alenabio Co., Xi'an, China) and mouse tissues. All mouse tissues were fixed in 4% paraformaldehyde for 24 hours. Bones were decalcified in 10% EDTA for 7 days at 4°C. Standard immunostaining techniques were used to prepare the sections for histology and immunohistochemistry staining. CD137, CD137L, Renilla luciferase, Ki67, and F4/80 were stained by using the following primary antibodies: anti-CD137 (abcam, Cambridge, UK), anti-CD137L (R&D Systems Inc., Minneapolis, MN, USA), anti-Renilla luciferase (Affinity Biosciences, Cincinnati, OH, USA), anti-Ki67 and F4/80 (abcam, Cambridge, UK) antibodies. The streptavidin-biotin detection system was applied and 3, 3′-diaminobenzidine (DAB) was used. The images were recorded by using Olympus BX51 fluorescent microscopy (Olympus, Tokyo, Japan).
F4 80
Manufactured by Abcam
Sourced in United Kingdom, United States, China, Germany, Japan, Canada
F4/80 is a cell surface glycoprotein that is commonly used as a marker for mature mouse macrophages. It is expressed on the surface of various macrophage populations, including those found in the liver, spleen, and peritoneal cavity.
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Lab products found in correlation
α sma, by Abcam (20 mentions)
Cd206, by Abcam (18 mentions)
Cd11b, by Abcam (14 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (11 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (10 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (9 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (8 mentions)
Tnf α, by Abcam (7 mentions)
Vimentin, by Abcam (6 mentions)
Foxp3, by Abcam (6 mentions)
Gapdh, by Abcam (5 mentions)
Cleaved caspase 3, by Cell Signaling Technology (5 mentions)
Cd163, by Abcam (5 mentions)
γ h2ax, by Abcam (5 mentions)
α sma, by Agilent Technologies (5 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (5 mentions)
Hoechst 33342, by Thermo Fisher Scientific (5 mentions)
Fibronectin, by Abcam (4 mentions)
α sma, by Merck Group (4 mentions)
Arginase 1, by Santa Cruz Biotechnology (4 mentions)
322 protocols using f4 80
1
Comprehensive Immunostaining Protocol for Tissue Analysis
2
Wound Healing Quantification Protocol
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Healing was assessed on a macroscopic level by imaging and quantifying eschar size 0, 2, 4, 6, 8, and 10 days post-wounding as previously described (Rebalka et al., 2015 (link)). Six micrometers paraffin embedded sections of skin, taken from the center of the wound, were sectioned and air-dried overnight. Masson's Trichrome staining (to measure wound area and assess granulation tissue), CD31 staining [used to assess vascular content; (1:50; Abcam)], and F4/80 staining [used to assess total macrophage content; number of F4/80 positive macrophages per unit area of muscle (1:100; Abcam)] were conducted using standard protocols.
3
Western Blotting and IHC Antibodies for Inflammation
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Antibodies used for western bloting included F4/80 (rat monoclonal, Abcam, ab16911; 1:200), CD11b (rabbit monoclonal, Abcam, ab133357; 1:1000), LY6C (rat monoclonal, Novus, NBP2-00441; 1:1000), MCP-1 (rabbit polyclonal, Cell Signaling Technology, #2029; 1:1000), NLRP3 (rabbit monoclonal, Cell Signaling Technology, #15101; 1:1000), pro-Caspase1 (rabbit monoclonal, Abcam, ab179515; 1:1000), GSDMD (rabbit monoclonal, Abcam, ab209845; 1:1000), IKK (rabbit monoclonal, Abcam, ab178870; 1:1000), phospho-IKK (rabbit monoclonal, Cell Signaling Technology, #2697; 1:1000), p65 (mouse monoclonal, Sabta cruz, sc-8008; 1:200), phospho-p65 (rabbit monoclonal, Cell Signaling Technology, #3033; 1:1000), IĸBα (mouse monoclonal, Cell Signaling Technology, #9246; 1:1000), phospho-IĸBα (mouse monoclonal, Cell Signaling Technology, #4814; 1:1000), GAPDH (mouse monoclonal, Abcam, ab8245; 1:8000), Goat Anti-Mouse IgG (Abcam, ab97023; 1:10000), Goat Anti-Rabbit IgG (Abcam, ab97051; 1:10000) and Goat Anti-Rat IgG (Abcam, ab97057; 1:10000). Antibodies used for immunohistochemistry and immunohistochemistry-frozen included F4/80 (rat monoclonal, Abcam, ab16911; 1:50) and LY6C (rat monoclonal, Novus, NBP2-00441; 1:200). LPS, IFN-γ, IL-4, IL-10 and M-CSF were purchased from PeproTech, ATP and nigericin were purchased from Med Chem Express.
4
Skeletal Muscle Histological Analysis
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Eight micrometers frozen TA muscle sections were stained via embryonic myosin heavy chain [eMHC; signal threshold settings used as the detection method (neat; Abcam; Cambridge, MA)] hematoxylin and eosin (H&E; used to quantify fiber area), F4/80 [used to assess total macrophage content; number of F4/80 positive macrophages per unit area of muscle (1:100; Abcam)] and alkaline phosphatase (used to assess vascular content; signal threshold settings used as the detection method) staining using standard protocols.
5
Histopathologic Evaluation of Prostate Cancer in Mice
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Formalin-fixed paraffin-embedded prostate tissue from each mouse was sectioned into three 4 μm sections, each separated by 50 μm, and stained with hematoxylin and eosin (H&E) for histopathologic diagnosis. Tumor tissue was graded as mouse PIN (mPIN) and adenocarcinoma according to current guidelines by a single reader blind to treatment arm, guided by a board-certified pathologist (GVT).9 (link) Subsequent serial sections were stained with antibodies against Ki67 (Thermo Scientific, Rockford, IL), TUNEL (ApopTag® Plus Peroxidase In Situ Apoptosis Kit; Millipore Inc.) and F4/80 (Abcam, Cambridge, MA) to assess proliferation, apoptosis and macrophage infiltration, respectively. Ki67 and TUNEL staining was conducted in both 3 and 6 month timepoints, whereas F4/80 staining was only carried out in the 6 month timepoint. Stained slides were scanned using an Aperio ScanScope and automated quantification of Ki67 and TUNEL expression was conducted blinded to dietary group using Definiens Tissue Studio® nuclear algorithm at the Translational Pathology Core, University of North Carolina at Chapel Hill. Manual quantification of F4/80 expression was conducted by a board-certified pathologist blinded to dietary group at the Translational Pathology Core, Cedars-Sinai Medical Center. Scores ranging from 1 – 3 were assigned based on the number of cells staining positive for F4/80.
6
Autophagosome Formation in Colonic Macrophages
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The formation of autophagosomes in colonic macrophages after TNBS induction with or without IL-33 administration was compared by immunofluorescence assay. Briefly, the sections were deparaffinized, rehydrated and washed in 1% PBS Tween. Then they were treated with 3% hydrogen peroxide, blocked with 5% bovine serum albumin (BSA) and incubated simultaneously with Beclin-1 (1:500, Sigma) and F4/80 (1:500, Abcam) overnight. After staining with the- secondary antibody, the slides were then counter-stained with DAPI for 5 min. Images were acquired by a fluorescence microscope (Olympus, Lake Success, NY). Settings for image acquisition were identical for control and experimental tissues.
7
Antibody selection for Western blot, IF, and FC
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The following rabbit anti-mouse antibodies purchased from Abcam were used for Western blotting: Nucleolin (ab22758), EGFR (ab52894), CD36 (ab133625), IFNγ (ab133566), TLR3 (ab62566), Fibrillarin (ab5821), Cytochrome C (ab133504), F4/80 (ab100790), β-actin (ab8227), and GAPDH (ab181602). The goat anti-rabbit lgG-Alexa Fluor 488 (Cat. # A27034) was purchased from Thermo Fisher for immunofluorescence. The FITC-F4/80 (Cat# 123108) antibodies purchased from Biolegend were used for flow cytometry.
8
Immunohistochemistry of Mouse Skin and Lungs
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Section (6-µm thickness) from paraffin-embedded mouse skin and lungs were deparaffinized and incubated overnight at 4 °C with mouse mAbs to CD3 (1:200; Nichirei Bioscience), F4/80 (1:1600; Abcam), and α-SMA (1:200; DAKO), followed by incubation with peroxidase-labeled secondary antibody (Nichirei BioScience) and color development with the aminoethyl carbazole system (Nichirei BioScience) [28 (link)]. Immunostained cells were counted in three high-power microscopic fields. Each section was examined independently by two investigators (T.C. and N.O.) in a blinded manner.
9
Inflammatory Cytokine Regulation Analysis
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Lipopolysaccharide was purchased from Sigma-Aldrich (St Louis, Ca). Recombinant mouse IL-4, recombinant mouse IL-6, recombinant mouse IL-1β, recombinant mouse TNF-α, recombinant mouse IL-10, and recombinant mouse TGF-β were purchased from R&D Systems (Minneapolis, MN). Tocilizumab, LY2109761, R-7050, Stattic, Bay 11-7082, and SIS3HCl were purchased from Selleck (Huston, USA). IL-1 receptor antagonist (IL1Ra) was purchased from Make Research Easy (Nanjing, China). Monoclonal mouse anti-β-actin (TA-09) was purchased from Zhongshan Golden Bridge Biotechnology (Beijing, China). Antibodies against OT, OTR, β-tubulin, CD206, F4-80, phospho-STAT3, STAT3, phospho-SMAD2, SMAD2, phospho-SMAD3, SMAD3, p65, and phospho-p65 were purchased from Abcam (Cambridge, UK) and Cell Signaling Technology (Danvers, MA). Secondary antibodies were purchased from Invitrogen Life Technology (Foster City, CA). Mouse ELISA kits were obtained from R&D Systems and CUSABIO (Wuhan, China). All reagents were analytical grade.
10
Histopathological Analysis of Liver Tissues
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Both the liver portion extracted at the time of hepatectomy and that collected at autopsy were processed for histopathological examination. Liver tissues were fixed in 10% formalin for 48 hours, routinely processed, and sliced into sections of 4 μm in thickness. For detection of liver fibrosis, sections were stained with picrosirius red (PSR; Sigma‐Aldrich), anti–α‐smooth muscle actin (α‐SMA) antibodies (AbCam) and anti‐desmin antibodies (Boehringer). For detection of macrophage infiltration, sections were stained with antibodies against CD68 (AbCam) and F4/80 (AbCam). For detection of liver proliferation, sections were stained with anti–proliferating cell nuclear antigen (PCNA) antibodies (Dako). Sections were also stained with hematoxylin‐eosin (H & E) and periodic acid–Schiff (both from Sigma‐Aldrich). After staining, specimens were photographed under a microscope (Olympus). Histological and immunohistochemical results were quantified using WinRoof 7.4 software (Mitani Corporation).
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