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Rat anti mouse f4 80 monoclonal antibody

Manufactured by Bio-Rad
Sourced in United States

The Rat anti-mouse F4/80 monoclonal antibody is a laboratory reagent used for the identification and characterization of mouse macrophages. It recognizes the F4/80 antigen, which is a glycoprotein expressed on the surface of mouse macrophages. This antibody can be used in various immunological techniques, such as flow cytometry, immunohistochemistry, and immunoprecipitation, to detect and study mouse macrophages.

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7 protocols using rat anti mouse f4 80 monoclonal antibody

1

Macrophage Visualization in Sciatic Nerve

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Under anesthesia with i.p. pentobarbital at 10 mg/kg following i.p. administration of a mixture of midazolam at 4 mg/kg and medetomidine at 0.3 mg/kg, mice were transcardially perfused with ice-cold saline in a volume of 20 mL and then with 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer in a volume of 50 mL. The sciatic nerve and L4-L6 DRG were removed and subjected to immunohistochemical detection of macrophages, as described previously [5 (link)]. Macrophages were visualized by immunofluorometric analysis, using a rat anti-mouse F4/80 monoclonal antibody (Bio-rad, Hercules, CA, USA) (1:1000 dilution), Cy3-conjgated goat anti-rat IgG antibody (Thermo Fisher Scientific, Carlsbad, CA, USA) (1:400 dilution), and 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) (Sigma-Aldrich) (1:2000 dilution), as reported previously [5 (link)]. The F4/80-positive cells were recognized by confocal laser fluorescence microscopy (FV10C-O, Olympus, Tokyo, Japan). The number of F4/80-positive cells were counted on each visual field.
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2

Immunohistochemical Analysis of Tumor Angiogenesis and Macrophages

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Frozen tumor sections were stained with hematoxylin and eosin (Wako Pure Chemical Industries Ltd.). Collagen was stained using a Masson’s trichrome staining kit (25088-100, Polysciences, Inc.). For immunochemical staining, frozen tumor sections were fixed in 10% formalin for 10 min at room temperature. After washing with 1 × TBS, sections were incubated with 0.3% H2O2 in methanol at room temperature, followed by washing in 1 × TBS. To inhibit non-specific staining, sections were incubated in serum-free protein block solution (X0909, Dako) for 15 min at room temperature. The sections were then incubated with primary antibodies: rat anti-mouse CD31 monoclonal antibody (BD557355, BD Biosciences, 1:100) or rat anti-mouse F4/80 monoclonal antibody (MCA497, Bio-Rad, 1:100). After washing with 1 × TBS, signal stain boost IHC detection reagent (anti-rabbit, Cell Signaling Technology) or rabbit anti-rat IgG H&L (horseradish peroxidase conjugated) (ab6734, Abcam, 1:500) was added, and sections were incubated at room temperature for 30 min. After reaction with diaminobenzidine (8059S, Cell Signaling Technology), sections were counterstained with hematoxylin. A BZ-X710 microscope (Keyence, Osaka, Japan) was used for histologic observation and evaluation.
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3

Immunohistochemical Analysis of Kidney Markers

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Paraffin-embedded kidney sections were deparaffinised and incubated with primary antibodies against collagen IV (dilution 1:1000, Abcam Ltd, Cambridge, USA), fibronectin (dilution 1:500, Abcam), or 8-hydroxy-2'–deoxyguanosine (8-OHdg) (dilution 1:200, Cell Signaling Technology, Beverly, USA) at 4°C overnight, followed by horseradish peroxidase anti-rabbit Envision system (Dako Tokyo, Japan) the following day.
Alternatively, frozen sections were incubated in the widely used markers of murine macrophage populations, rat anti-mouse F4/80 monoclonal antibody (dilution 1:100, ABD Serotec, USA) or rat anti-mouse CD68 antibody (dilution 1:100, ABD Serotec) for one hour. Thereafter, they were incubated with a secondary HRP labelled goat anti-rat antibody for 30 min (ABD Serotec, dilution 1:200).
Antigen-antibody reactions were visualized with 3.3diaminobenzidine tetrahydrochloride (Dako) and counterstained. The tissue specimens were examined by light microscopy using the Olympus photomicroscope. For fibronectin, collagen IV, 8-OHdg, CD68, and F4/80, six consecutive non-overlapping fields from each section of renal cortex were photographed under high magnification. Stained areas were quantified using Image J software (NIH, UK).
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4

Histological Analysis of Spinal Cord in EAE Mice

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Histological analysis was performed as previously described (26 (link), 27 (link)). Mice with peak EAE were anesthetized and perfused transcardially with 4% paraformaldehyde in 0.1 M PBS. Lumbosacral spinal cords were immediately removed, post-fixed in 4% paraformaldehyde, and embedded in paraffin or OCT compound (Sakura Finetek Japan, Tokyo, Japan). Eight-micron-thick paraffin sections were stained with hematoxylin and eosin. Stained sections were analyzed on a NanoZoomer 2.0-RS slide scanner (Hamamatsu Photonics, Hamamatsu, Japan). For immunofluorescence staining, the frozen sections (20 μm) were permeabilized with 0.1% Triton X-100 in PBS for 20 min, blocked with 5% bovine serum for 30 min, and then incubated overnight with rat anti-mouse F4/80 monoclonal antibody (CI:A3-1; 1:1,000, AbD Serotec, Raleigh, NC, USA), rabbit anti-mouse iNOS polyclonal antibodies (1:5,000, Merck Millipore), and goat anti-mouse Arginase-1 polyclonal antibodies (1:200, Proteintech) followed by incubation with Alexa Fluor 488 or 546-conjugated secondary antibodies (Life Technologies, Carlsbad, CA, USA). The stained cells were analyzed by examining six random fields per section using a deconvolution fluorescence microscope system (BZ-X800, Keyence, Osaka, Japan). The data were collected from three animals per group.
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5

Histological Analysis of Liver Fibrosis

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Liver tissue was fixed in 10 % (v/v) neutral buffered formalin solution, dehydrated with ethanol, cleared in xylene and embedded in paraffin. Then the paraffin-embedded blocks were cut into sections approximately 5 µm thick. After removal of paraffin with xylene, sections were stained with haematoxylin and eosin (Merck)(30 ) for morphological analysis, or were stained with Sirius red (Sigma-Aldrich) and counterstained with fast green (Wako) for determination of the area of fibrosis(31 (link)). Immunohistochemical staining of hepatic monocytes/macrophages was performed by using sections of formalin-fixed, paraffin-embedded liver tissue as described previously(32 (link),33 (link)). Briefly, after removal of paraffin, sections were incubated with a rat anti-mouse F4/80 monoclonal antibody (Serotec), followed by incubation with a biotinylated rabbit anti-rat IgG antibody (Dako) and peroxidase-conjugated streptavidin (Dako). Then colour was developed with diaminobenzidine tetrahydrochloride (Dojindo) and the sections were counterstained with haematoxylin. Images were acquired with an Olympus DP73 digital camera (Olympus IX-73; Olympus) under an inverted microscope (original magnification, ×140). The F4/80-positive area and Sirius red-positive area were quantified as a percentage of the total tissue area by using cellSens Dimension Olympus 1.15 software (Olympus).
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6

Mouse Macrophage F4/80 Immunohistochemistry

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Mouse macrophage specific F4/80 staining was performed as described by Blom et al.20 (link) Briefly, sections were deparaffinized, rinsed with PBS, and then incubated in 3% H2O2 in methanol for 30 min at room temperature to deactivate endogenous peroxidase. After blocking with normal rabbit serum (Vector Laboratories, CA) for 30 min, sections were incubated with rat anti-mouse monoclonal F4/80 antibody (1:2000 dilution: AbD Serotec, Raleigh, NC) at 4°C overnight. After extensive washing with PBS, signals were visualized by the Vectastain ABC kit (Vector Laboratories). Therefore, sections were counterstained using hematoxylin.
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7

Mouse Model of Inflammation

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Animal care and all experiments were conducted in accordance with the guideline of the Animal Committee of Tokyo Medical and Dental University. C57BL/6J mice were purchased from ORIENTAL YEAST Co., Ltd. (Tokyo, Japan). Mice were housed under a 12-hour light-dark cycle and allowed food and water ad libitum. λ-Carrageenan was purchased from Sigma-Aldrich (St. Louis, MO). Recombinant mouse follistatin was purchased from R&D Systems Inc. (Minneapolis, MN). Rat anti-mouse monoclonal F4/80 antibody was purchased from AbD Serotec (Oxford, UK).
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