Liver sections (7 µm thick) were fixed in cold acetone, and stained with hematoxylin/eosin staining. For triple immunohistochemical analyses, sections were reacted with rat anti-mouse CD31 (1:100, Dako), rabbit anti-mouse Desmin (1:200, Dako), and mouse anti-human α-SMA (1:50, Sigma-Aldrich) followed by the appropriate fluorescent secondary antibodies (n = 72). Serial sections were incubated with either rat anti-F4/80 (1:100, BIO-RAD) (n = 36) or mouse anti-Ki67 (1:200; Sigma-Aldrich) (n = 36), followed by appropriate fluorescent secondary antibodies. Irrelevant appropriate immunoglobulins were used as negative controls. Metastases were microphotographed using a Zeiss Axioskop fluorescence microscope under the same conditions. Specific immunostaining was quantified using FIJI-ImageJ software. Results were expressed as the percentage of specifically colored tissue area relative to the whole micrometastatic foci area.
Rat anti f4 80
Manufactured by Bio-Rad
Sourced in United States, United Kingdom
The Rat anti-F4/80 is a laboratory reagent used for the identification and characterization of macrophages in rat samples. It specifically binds to the F4/80 antigen, which is expressed on the surface of mature murine macrophages. This product can be used in various applications, such as flow cytometry, immunohistochemistry, and immunofluorescence, to detect and study macrophage populations in rat biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Bz x710, by Keyence (3 mentions)
Rat anti ptprc cd45r, by Aviva Systems Biology (3 mentions)
Anti cd4, by Cell Signaling Technology (2 mentions)
Anti cd8, by Cell Signaling Technology (2 mentions)
Rabbit anti syndecan 1, by Bioss Antibodies (2 mentions)
Rabbit anti inos, by Abcam (2 mentions)
Goat anti opn, by R&D Systems (2 mentions)
Mouse anti pcna, by Cell Signaling Technology (2 mentions)
Horseradish peroxidase conjugated secondary antibody, by Santa Cruz Biotechnology (2 mentions)
Mouse anti wt1 antibody, by Santa Cruz Biotechnology (2 mentions)
Rabbit anti mouse collagen 4, by Bio-Rad (2 mentions)
Rabbit anti n tyr antibody, by Santa Cruz Biotechnology (2 mentions)
Sigma fast dab chromogenic tablets, by Merck Group (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Rabbit anti p c fms csf 1r tyr723 and nitrotyrosine, by Santa Cruz Biotechnology (2 mentions)
Goat anti csf 1, by Santa Cruz Biotechnology (2 mentions)
Mouse anti kim 1, by R&D Systems (2 mentions)
Rabbit anti hmgb1, by Cell Signaling Technology (2 mentions)
Rabbit anti human fibronectin, by Merck Group (2 mentions)
Axioskop fluorescence microscope, by Zeiss (1 mentions)
32 protocols using rat anti f4 80
1
Immunohistochemical Profiling of Liver Metastases
2
Multicolor Immunofluorescence Staining Protocol
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Chicken anti-GFP antibody (Abcam, ab13970, 1:500), Rabbit anti-Prospc (Millipore, ab3786, 1:500), Rat anti-Ki67 (ebioscience, 14-5698-82, 1:200), Rat anti-F4/80 (BioRad, MCA497GA, 1:200), APC anti-mouse CD326 (BioLegend, catalog#118214), PE/Cyanine7 anti-mouse CD45 (BioLegend, catalog#103114), PE/Cyanine7 anti-mouse CD31 (BioLegend, catalog#102418), PE anti-mouse F4/80 (BioLegend, catalog#123110), APC anti-mouse CD11c (BioLegend, catalog#117310), PerCP-Cyanine5.5 anti-human/mouse CD11b (Tonbo, catalog#65-0112), PE anti-mouse FOXP3 (BD biosciences, catalog#560408), APC anti-mouse CD4 (BD biosciences, catalog#553051), FITC Mouse IgG1 isotype control (BD biosciences, catalog#555748), PE Mouse IgG1 isotype control (BD biosciences, catalog#555749), APC Mouse IgG1 isotype control (BD biosciences, catalog#555751), FITC anti-human CD90 (BD biosciences, catalog#555595), PE anti-human CD73 (BD biosciences, catalog#550257), APC anti-human CD105 (BD biosciences, catalog#562408), FITC anti-human CD45 (BD biosciences, catalog#555482), PE anti-human CD34 (BD biosciences, 550761), Alexa Fluor 488 Donkey anti Chicken (Jackson Immuno Research, catalog#703-545-155), Alexa Fluor Cy3 Donkey anti Rat (Jackson Immuno Research, catalog#712-165-153), and Alexa Fluor 488 Donkey anti Rabbit (Jackson Immuno Research, catalog#711-545-152).
3
Immunohistochemical Analysis of Hypoxia Markers
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We used paraformaldehyde-fixed, paraffin-embedded samples of one microgram tissue thickness as previously described [36 (link)]. In essence, heat-induced epitope retrieval was performed in a citrate buffer at pH 6.0 followed by diverse blocking steps and detection with the ABC Vectastain kit (Maravai Life Sciences, San Diego, USA). Immunohistochemistry for HIF-1α was done with the Dako CSAII-Kit (Dako Agilent, Santa Clara, USA). As primary antibodies we used rabbit-anti-HIF-1α (Cayman Chemical, Ann Arbor, USA Cat# 10006421, RRID:AB_409037) at a dilution of 1:10.000, rat-anti-F4/80 (Bio-Rad, Hercules, USA Cat# MCA497, RRID:AB_2098196) at a dilution of 1:200, rat-anti-VEGF (BioLegend, San Diego, USA Cat# 512901, RRID:AB_2212504) at a dilution of 1:300, rabbit-anti-PHD2 (novus Biologicals, Cat# 100–137, RRID:AB_350074 at a dilution of 1:200. For the detection of F4/80 and VEGF, biotinylated anti-rat antibody (Santa Cruz Biotechnology, Dallas, USA Cat# sc-2041, RRID:AB_631752) and for the detection of PHD2, biotinylated anti-rat antibody (Santa Cruz Biotechnology, Dallas, USA, Cat# sc-2040, RRID:AB_631743) was used as the secondary antibody (1:200).
4
Comprehensive Immunohistochemical Analysis of Tissues
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Each joint and lymph node was fixed in 4% paraformaldehyde/PBS, decalcified in 2.5% EDTA, and stored in 7% sucrose at 4 °C for one month. Paraffin Sects. (2 μm) were stained with hematoxylin and eosin (H&E; Muto, Tokyo, Japan) for immunohistochemical analysis. The sections were stained with the following primary antibodies: rat anti-F4/80 (Bio-Rad), rabbit anti-CD3 (Genemed Biotechnologies, South San Francisco, CA, USA), rat anti-PTPRC/CD45R (Aviva Systems Biology, San Diego, CA, USA), rabbit anti-syndecan 1 (Bioss Antibodies, Woburn, MA, USA), anti-CD4, and anti-CD8 (Cell Signaling Technology, Danvers, MA, USA). Histofine simple stain mouse MAX-PO secondary antibodies and the Opal multiplex fluorescent immunohistochemistry system (Akoya Biosciences, Marlborough, MA, USA) were used according to the manufacturers’ protocols. All images were captured using a fluorescence microscope (BZ-X710; Keyence, Osaka, Japan).
5
Immunocytochemical Staining of RAGE, F4/80, and iNOS
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For immunocytochemistry, cells were fixed in 4% PFA for 30 min. After blocking in 3% BSA without Triton X-100 for 30 min, the cells were stained with the following primary antibodies: rabbit anti-RAGE antibody (1:200, Proteintech), rat anti-F4/80 (1:200, Bio-Rad), and rabbit anti-iNOS (1:200, Abcam). Cells were then incubated with the corresponding secondary antibodies. Sections were examined and photographed under a confocal microscope (LSM 800, Zeiss).
6
Quantifying Macrophage Subsets in Tissue Sections
7
Comprehensive Lung Tissue Analysis
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The mice were euthanised and the lungs harvested and fixed with 4% paraformaldehyde diluted in PBS. Paraffin sections (2 μm) were stained with haematoxylin–eosin (H&E), Masson's trichrome and the following primary antibodies for immunohistochemistry: rabbit anti-E-cadherin, rabbit anti-α SMA, rabbit anti-vimentin, rabbit anti-MMP7 (Cell Signalling Technology, Danvers, MA, USA), rabbit anti-SP-C (Hycult Biotech, Uden, Netherlands), rat anti-Podoplanin (MBL, Aichi, Japan), rabbit anti-collagen I (Novus Biologicals, Littleton, CO, USA), rat anti-F4/80 (Bio-Rad Laboratories, Hercules, CA, USA), rabbit anti-CD3 (Genemed Biotechnologies, San Franciso, CA, USA), rat anti-PTPRC/CD45R (Aviva Systems Biology, San Diego, CA, USA), rabbit anti-arginase I (GeneTex, Irvine, CA, USA), mouse anti-peptidyl-citrulline (F95) and rabbit anti-S100A4 (Millipore, Burlington, MA, USA) and mouse anti-Laminin γ2 N-terminal fragment (γ2pf; Funakoshi, Tokyo, Japan). Histofine Simple Stain Mouse MAX-PO secondary antibodies (Nichirei, Tokyo, Japan) and the Opal 4-colour Fluorescent IHC kit (PerkinElmer, Waltham, MA, USA) were used according to the manufacturer's protocol. All images were captured by fluorescence microscopy (BZ-X710, Keyence) and analysed by hybrid cell count (Keyence, Osaka, Japan) or Fiji.
8
Immunofluorescence Staining of Frozen Tissues
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Frozen sections (5 μm) were fixed with acetone for 10 min, penetrated by 10% Triton for 10 min, and then blocked with 5% goat serum for 1 h. The tissues were incubated overnight with primary antibodies. Rabbit anti-fibronectin (1:200, Abcam), rabbit anti-collagen I (1:200, Abcam), mouse anti-α smooth muscle actin (1:200, Sigma), mouseanti-CD45 (1:100, BD), rabbit anti-α-SMA (1:100, Abcam), rat anti-F4/80 (1:100, Bio-Rad), rabbit anti-α-SMA (1:100, Abcam), rat anti-CD206 (1:100, Bio-Rad), and rabbit anti-α-SMA (1:100, Abcam) at 4 °C. The secondary antibodies of Alexa Fluor 488 goat anti-rabbit (1:400, Abcam), Alexa Fluor 647 goat anti-mouse (1:400, Abcam) and Alexa Fluor 647 goat anti-rat (1:400, Abcam) we reselected to incubate at room temperature for 1 h. Dye the nucleus with DAPI. The images were taken by fluorescence microscope equipped with digital camera (Olympus, Japan) orconfocal laser scanning microscope (Zwiss, Germany). Measurement of the fluorescence staining area was performed using ImageJ software.
9
Histological Analysis of Renal Fibrosis
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Renal tissues were fixed in 10% formalin for 20 hours and embedded in paraffin. Four micrometer sections were cut for all stainings. All slides were quantified in a blinded fashion. Periodic acid-Schiff reagents after diastase digestion (PAS-D) were performed according to standard diagnostic procedures. Mesangial matrix expansion was semi-quantitatively scored in 15 glomeruli by a pathologist on a scale of 0 to 3 (0 = absent, 1 = mild, 2 = moderate, 3 = severe). Goat anti-collagen IV (polyclonal, Merck Millipore, Amsterdam, The Netherlands), rat anti-CD31/PECAM (Clone SZ31, Dianova, Sankt Augustin, Germany), rat anti-F4/80 (Clone CL:A3-1, Bio-Rad, Hercules, CA, USA), rabbit anti-α-SMA (Clone 1A4, DAKO, Santa Clara, CA, USA), hamster anti-ICAM-1 (Clone 3E2, BD Pharmingen, San Jose, CA, USA), rabbit anti-iNOS (polyclonal, Abcam, Cambridge, UK), podocin (polyclonal, Sigma-Aldrich, Zwijndrecht, The Netherlands) and rabbit anti-cleaved caspase-3 (Clone Asp175, Cell signaling Technology, Beverly, MA, USA) antibodies were used for immunostainings. The cortical positive area was determined in 10 high power fields and glomerular positive area was determined in 10-15 glomeruli using image analysis with Fiji software (ImageJ, https://fiji.sc/).
10
Immunohistochemical Analysis of Joint and Lymph Node Tissues
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Each joint and lymph node was xed in 4% paraformaldehyde/PBS, decalci ed in 2.5% EDTA, and stored in 7% sucrose at 4°C for one month. Para n sections (2 mm) were stained with hematoxylin and eosin (H&E; Muto, Tokyo, Japan) for immunohistochemical analysis. The sections were stained with the following primary antibodies: rat anti-F4/80 (Bio-Rad), rabbit anti-CD3 (Genemed Biotechnologies, South San Francisco, CA, USA), rat anti-PTPRC/CD45R (Aviva Systems Biology, San Diego, CA, USA), rabbit antisyndecan 1 (Bioss Antibodies, Woburn, MA, USA), anti-CD4, and anti-CD8 (Cell Signaling Technology, Danvers, MA, USA). Histo ne simple stain mouse MAX-PO secondary antibodies and the Opal multiplex uorescent immunohistochemistry system (Akoya Biosciences, Marlborough, MA, USA) were used according to the manufacturers' protocols. All images were captured using a uorescence microscope (BZ-X710; Keyence, Osaka, Japan).
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