Urinary albumin and creatinine were determined by using mouse albumin ELISA and creatinine companion kits (Philadelphia, PA USA) following manufacturer’s protocols. Serum levels of creatinine were determined by using a kit from Biovision (Milpitas, CA). For immunohistochemical staining, formalin-fixed and paraffin-embedded renal tissues were cut into 4- to 5-μm sections. The slides were stained with anti-collagen IV (Cell Signaling) or F4/80 antibody (Serotec) using VECTASTAIN Elite ABC system (Vector Lab) as described previously [25 (link)]. For immunofluorescence staining: frozen kidney samples were sectioned and fixed in cold acetone. The slides were stained with anti-WT1 antibody (Novus) and then with fluorescence conjugated secondary antibodies (Life Technologies). After washing, slides were incubated with DAPI for 5 minutes and coverslipped. In addition, kidney electron microscopy (EM) was performed by using the service from the Imaging Center at University of Kentucky.
F4 80 antibody
Manufactured by Bio-Rad
Sourced in United Kingdom, United States, Germany
The F4/80 antibody is a laboratory research tool used to detect and study the F4/80 antigen, which is expressed on the surface of mouse macrophages. It can be used in various immunological techniques, such as flow cytometry and immunohistochemistry, to identify and characterize macrophage populations in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (3 mentions)
Doxycycline, by Bio-Serv (2 mentions)
Fluorescence conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Vectastain elite abc system, by Vector Laboratories (1 mentions)
Anti collagen 4, by Cell Signaling Technology (1 mentions)
Anti sirt1 antibody, by Abcam (1 mentions)
F4 80 antibody, by Abcam (1 mentions)
Anti ho 1 antibody, by Abcam (1 mentions)
Bz 9000 fluorescence microscope, by Keyence (1 mentions)
Lsm 710 meta, by Zeiss (1 mentions)
Alexa fluor 488 donkey anti mouse, by Thermo Fisher Scientific (1 mentions)
Keratin 14, by Cell Signaling Technology (1 mentions)
Donkey anti rat alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
Pdgfra, by R&D Systems (1 mentions)
Perilipin a, by Cell Signaling Technology (1 mentions)
Alexa fluor 594 donkey anti goat, by Thermo Fisher Scientific (1 mentions)
Hmgb1, by Cell Signaling Technology (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
St5020, by Leica (1 mentions)
α smooth muscle actin antibody, by Abcam (1 mentions)
20 protocols using f4 80 antibody
1
Comprehensive Renal Histological and Functional Analyses
2
Characterization of Transplanted Tissues in Lean and Obese Mice
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The pancreas, adipose tissue, thymus, and livers of the lean and db mice were removed 2 months after transplantation. The pancreata were stained with polyclonal guinea pig anti-swine insulin antibody (N1542, Dako Cytomation, CA) and monoclonal rabbit anti-human glucagon (8233s, Cell Signaling Technology, Massachusetts). The adipose tissue was stained with F4/80 antibody (AbD Serotec, Oxford, UK). The livers were stained with hematoxylin and eosin (HE), anti-Sirt1 antibody (AbcamⓇ), F4/80 antibody, and anti-HO-1 antibody (AbcamⓇ). The thymi were stained with anti-cytokeratin 5 (CK5) antibody (AbcamⓇ), Sirt1 antibody, and HO-1 antibody. The bone marrow was stained with anti-HO-1 antibody. The stained sections were examined under an Olympus microscope (Olympus, Japan). HO-1 and Sirt1-positive cells in the liver of each group were calculated using software (Image-pro plus) for Windows.
3
Tetracycline-Regulated BP1 Transgenic Mice
4
Tetracycline-Regulated BP1 Transgenic Mice
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The generation of the tetracycline-regulated conditional BP1 transgenic mice, and the impact of BP1 on angiogenesis and wound healing, were previously described by Tassi et al. [19 (link)]. BP1 transgene (Tg) expression is silenced by feeding these mice a diet supplemented with an orally available tetracycline (e.g. doxycycline) from Bio-Serv (Frenchtown NH) (BP1 OFF) and induced by a switch to regular mouse chow that lacks tetracycline (BP1 ON), typically for 2 weeks before the experiments. The dorsal skin of BP1 OFF and BP1 ON animals was injured by a full-thickness dermal biopsy punch (3 mm diameter) under anesthesia, as previously described [19 (link)]. Histological sections were then screened for the presence / absence of KS-like lesions. Immunohistochemistry studies were done to detect the infiltration of macrophages using the F4/80 antibody (Serotec, Raleigh, NC) as described by Tassi et al. [19 (link)]. Animals experiments were reviewed and approved by the Institutional Animal Care and Use Committee at both institutions, Children’s National Medical Center, and Lombardi Cancer Center, at Georgetown University, Washington DC.
5
Quantifying Intestinal Inflammatory Monocytes
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The whole small intestine was explanted, and 1 cm of the aboral end was cut and frozen in kryoTec freezing medium (Kryotec-Kryosafe GmbH, Hamburg, Germany). Five-µm slices were cut and fixed with acetone. Staining of inflammatory monocytes was performed with F4/80 antibody (AbD Serotec, Kidlington, UK) and secondary antibody goat anti-rat A647 (abcam plc. Cambridge, UK). DAPI (Thermo Fisher Scientific Inc) was used to counterstain cell nuclei. For histological analysis, images were recorded as 24-bit TIFF files on a Keyence BZ-9000® fluorescence microscope (Keyence Corporation, Osaka, Japan) with 20 × magnification, then saved using the BZ-II Image Viewer (Ver. 1.41, Keyence Corporation) and Analyzer (Ver. 1.42, Keyence Corporation). Cell segmentation was determined using a "positive cell detection" algorithm in QuPath (0.2.0-m4, University of Edinburgh, Scotland) with cell categorization by cytoplasm58 (link). Data were compiled using jupyterlab (1.2.5) with the following packages: Python (Ver. 3.6.8), Numpy (Ver. 1.18.1) and Pandas (Ver. 0.24.0). Each biological replicate was calculated by the mean of three technical replicates.
6
Histological Analysis of Mouse Skin
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Dorsal skin of mice was processed for histological sections, and 5-μm-thick paraffin sections were subjected to H/E staining or immunohistochemical analysis. The antibodies used for immunochemical detection were anti-15-lipoxygenase-1 antibody (Abcam), Perilipin A (Cell Signaling), Keratin 14 (Cell Signaling), Keratin 15 (Cell Signaling), PDGFRA (R&D systems), smooth muscle actin-FICT (Sigma), HMGB1 (Cell Signaling), tdTomato (Clonetech), Ly6C (Biolegend) and F4/80 antibody (AbD Serotec). The secondary antibodies used were donkey anti-mouse-Alexa Fluor 488, donkey anti-rabbit-Alexa Fluor 594/Alexa Fluor 488, donkey anti-goat-Alexa Fluor 594, and donkey anti-rat-Alexa Fluor 594 (ThermoFisher Scientific, Molecular Probes). The omission of primary antibody or normal rabbit, rat, goat, or mouse IgG controls (Santa Cruz Biotechnology) was used as a negative control. DAPI (Sigma) was used for nuclear counterstaining. Cells were imaged on a Zeiss confocal laser-scanning microscope (LSM 710 META, Zeiss, Jena, Germany).
7
Macrophage/Monocyte Detection and Fat Cell Morphometry
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For detection of macrophages/monocytes, an F4/80+ antibody (product code: MCA497G, AbD Serotec, Düsseldorf, Germany) was used for mice samples, a CD68-monoclonal antibody (Clone EBM11, Dako, Denmark) was used for human samples. Visualization of the complex was done using 3,3′-diaminobenzidene for 5 min. Negative controls were used by omitting the primary antibody. Morphometry of individual fat cells was assessed using digital image analysis. Microscopic images were digitized in 24-bit RGB (specimen pixel size 1.28×1.28 μm2). Recognition of fat cells was initially performed by applying a region-growing algorithm on manually indicated seed points, and minimum Feret diameter was calculated.
8
Immunohistochemical Detection of F4/80 in WAT
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For detection of F4/80, 5 μm paraffin-imbedded WAT sections were fixed and processed for immunohistochemistry using the avidin-biotin peroxidase complex (ABC, ThermoFisher, Waltham, MA) method and diaminobenzidine (DAB)-nickel reactions (Abcam, Cambridge, UK) as previously described [64 (link)]. Briefly, sections were de-paraffinized and dehydrated. Sections were incubated sequentially with 3% H2O2 to block peroxidase, F4/80 antibody (AbD SeroTec, Kidlinton, UK), then followed by secondary antibody (anti-rat) and ABC solution. After the DAB-nickel reaction, the sections were counterstained with 0.1% neutral red solution, and analyzed using a light microscope (Olympus DX 51, Olympus, Tokyo) and a charge-coupled device camera (Olympus DP12).
9
Quantitative Macrophage Analysis in Tissues
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All collected tissues were fixed in 10% Neutral Buffered Formalin (NBF) for 24 h, processed to paraffin blocks, and cut into 4 micron sections. The sections were dried overnight in a 37 °C oven, followed by 1 hour incubation in a 60 °C oven prior to deparaffinization. Deparaffinization and H&E staining (Surgipath, Buffalo Grove, IL, USA) were performed on an automated multistainer (Leica ST 5020, Buffalo Grove, IL, USA). The F4/80 antibody, an IgG2b affinity purified rat monoclonal antibody, was purchased from AbD Serotec. The α smooth muscle actin antibody was purchased from Abcam. Morphometric analysis was performed using a Scan Scope XT (Aperio) and both Image Scope (Aperio) and Indica Lab (Indica Lab) software. For each animal, nine 3.6 × 105 μm2 areas were evaluated for the percent F4/80 positive staining per area of tissue.
10
Immunohistochemical Analysis of TBI in Mice
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To assess the effects of CN-105 on inflammation, neuronal injury and neuronal loss, immunohistochemical(IHC) staining was performed using the F4/80 antibody(a marker for mature microglia and macrophages; rat monoclonal, 1:10,000; Serotec, Raleigh, NC) and the Fluoro-Jade B stain(a marker of degenerating neurons; Histo-Chem Inc. Jefferson, AR) on days 10 and 1, respectively, after TBI. IHC was performed on separate cohorts of mice from those used in neurobehavioral tests. As previously described82 (link), mice were anesthetized, euthanized, and perfused with 30 ml phosphate-buffered saline(PBS) via transcardiac puncture. The brains were then immersed in buffered formalin overnight and saturated with 30% sucrose in buffer. Sagittal sections(40 μm) were sliced on a freezing microtome and collected in cryoprotectant solution. For histological assessment the following were use: Secondary antibody, biotinylated goat anti-mouse IgG(1:3,000), ABC, and DAB all from Vector Laboratories, Inc., Burlingame, CA. and Gill’s Hematoxylin, Fisher Scientific, Fair Lawn, NJ.
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