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Rat anti mouse f4 80

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The Rat anti-mouse F4/80 is a monoclonal antibody that recognizes the mouse F4/80 antigen, a cell surface glycoprotein expressed on mature macrophages. This antibody is a useful tool for the identification and analysis of macrophages in various mouse tissues and models.

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Lab products found in correlation

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46 protocols using rat anti mouse f4 80

1

Renal Histopathology and Fibrosis

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Tubular injuries were assessed in periodic acid-Schiff (PAS)-stained kidney sections. For the immunohistochemical staining, we used rat anti-mouse F4/80 (Serotec, Kidlington, UK) and TGF-β1 (Abcam) antibodies. A total of 8–10 high power fields (HPFs) were captured, and the mean number of positive cells was compared between the groups. Renal fibrosis was assessed by Masson trichrome staining, and the area of fibrosis was expressed as the percentage of the blue-stained area in the renal cortex and the outer medulla.
Kim M.G., Kim S.C., Ko Y.S., Lee H.Y., Jo S.K, & Cho W. (2015). The Role of M2 Macrophages in the Progression of Chronic Kidney Disease following Acute Kidney Injury. PLoS ONE, 10(12), e0143961.
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2

Immunofluorescence Analysis of Cardiac Tissue

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Immunofluorescence was performed on 5-μm thick formalin-fixed and OCT-embedded heart sections. Briefly, formalin-fixed paraffin embedded sections were deparaffinized, followed by antigen retrieval and blocking with blocking buffer (1% BSA in 1× PBS) for 1 h [20 (link)]. Similarly, OCT-embedded sections were fixed with 4% paraformaldehyde for 20 min and rehydrated in 1× PBS for 30 min. Sections were then incubated with primary antibody against rat anti-mouse neutrophil (Serotec), rat anti-mouse F4/80 (Serotec), mouse anti-nitrotyrosine (anti-NT) (Santa Cruz Biotechnology), mouse anti-4-hydroxynonenal (anti-4-HNE) (Abcam), overnight in a humidified chamber at 4°C. Sections were incubated with different fluorophore-conjugated secondary antibodies (Invitrogen, U.S.A.) as described recently [20 (link)].
Das S.K., Zhabyeyev P., Basu R., Patel V.B., Dyck J.R., Kassiri Z, & Oudit G.Y. (2018). Advanced iron-overload cardiomyopathy in a genetic murine model is rescued by resveratrol therapy. Bioscience Reports, 38(1), BSR20171302.
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3

Quantitative Histological and Biochemical Assessment of Liver Injury

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Hematoxylin-eosin and Sirius red staining were performed according to standard protocols. For immunohistochemical analysis, liver specimens were fixed in 10% buffered formalin or 4% paraformaldehyde and incubated with rat anti-mouse F4/80 (Clone: A3-1, AbD Serotec, Raleigh, NC, USA), rat anti-mouse CD68 (Clone: FA-11, AbD Serotec), goat anti-mouse Gpnmb (Clone: #297310, R&D Systems, Minneapolis, MN, USA), and monoclonal antibody against α-SMA (Clone: E184, Merck Millipore, Billerica, MA, USA). For immunofluorescent staining, paraffin sections were incubated with rat anti-mouse CD68 (Clone: FA-11, AbD Serotec) and goat anti-mouse Gpnmb (Clone: #297310, R&D Systems). These samples then imaged with fluorescent microscopy. The terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling assay (Promega, Madison, WI, USA) was performed on paraffin liver sections according to the manufacturer’s instructions. All samples undergoing immunofluorescent staining were labeled with ProLongR Gold Antifade Mountant with DAPI (Thermo Fisher Scientific Inc., Grand Island, NY, USA). Serum levels of alanine aminotransferase (ALT) were measured with a commercial kit (SRL, Tokyo, Japan).
Kumagai K., Tabu K., Sasaki F., Takami Y., Morinaga Y., Mawatari S., Hashimoto S., Tanoue S., Kanmura S., Tamai T., Moriuchi A., Uto H., Tsubouchi H, & Ido A. (2015). Glycoprotein Nonmetastatic Melanoma B (Gpnmb)-Positive Macrophages Contribute to the Balance between Fibrosis and Fibrolysis during the Repair of Acute Liver Injury in Mice. PLoS ONE, 10(11), e0143413.
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4

Immunohistochemical Staining of Tissue

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The tissues collected at the time of sacrifice were fixed with buffered 20% formalin (Sumitani Shoten Co., Ltd, Japan) and embedded in paraffin. Histological and immunohistochemical staining of tissue sections using rat anti-mouse F4/80 (AbD Serotec, UK) or rabbit anti-SIRPα (Abcam, UK) was performed as previously described [2 (link)].
Morimoto A., Uchida K., Chambers J.K., Sato K., Hong J., Sanjoba C., Matsumoto Y., Yamagishi J, & Goto Y. (2019). Hemophagocytosis induced by Leishmania donovani infection is beneficial to parasite survival within macrophages. PLoS Neglected Tropical Diseases, 13(11), e0007816.
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5

Immunohistochemical Analysis of Inflammation Markers

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Staining for CD68 and F4/80 was performed on acetone-fixed frozen sections (7 µm) and endogenous biotin was blocked using a biotin blocker system (DAKO, Carpinteria, CA). For TLR4 and WT1 detection, formalin-fixed sections were deparaffinized and boiled in 10 mM sodium citrate buffer (pH 6.0). Sections were then incubated with 10% normal horse serum followed by 60-minute incubation with primary antibodies: a rat anti-mouse CD68 antibody (ABD Serotec Inc., Oxford, UK), a rat anti-mouse F4/80 (ABD Serotec Inc.), rabbit anti-mouse-TLR4 antibody (Invitrogen, Catalogue No. 48-2300) for glomerular [22] (link), goat anti-mouse-TLR4 antibody (Santa Cruz Biotechnology, Catalogue No sc-12511) for tubular [8] (link), rabbit anti-WT1 antibody (Abcam, Cambridge, UK), or concentration-matched IgG as an isotype negative control. The sections were exposed to H2O2 and then incubated with biotinylated anti-rat IgG or anti-rabbit IgG (BD Biosciences, Pharmingen), or anti-goat IgG (Vector Laboratories Inc). A Vector stain ABC kit (Vector Laboratories Inc) was applied to the tissue followed by DAB solution (DAKO). Immunostaining for α-SMA was performed on formalin-fixed paraffin sections using Dako ARK Peroxidase for Mouse Primary Antibodies (DAKO) according to the manufacturer's instructions.
Ma J., Chadban S.J., Zhao C.Y., Chen X., Kwan T., Panchapakesan U., Pollock C.A, & Wu H. (2014). TLR4 Activation Promotes Podocyte Injury and Interstitial Fibrosis in Diabetic Nephropathy. PLoS ONE, 9(5), e97985.
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6

Histological and Immunohistochemical Analysis of Gut Samples

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Gut paraffin sections were stained with hematoxylin and eosin (H&E), and analyzed under light microscopy. Gut cryosections (2 μm) were prepared for immunohistochemistry and processed as previously described with the following primary antibodies rat anti-mouse GR-1, rat anti-mouse F4/80 and anti-CD3 (Clone: 145-2C11) (AbD Serotec, Oxford, UK) [23 (link)]. Cytospin preparations of 105 cells were prepared (10 min,600 g) and stained according to May-Gruenwald-Giemsa and analyzed with an Olympus CKX41 light microscope (Shinjuku, Tokyo, Japan) [24 (link)].
Yu Y., Feng X., Vieten G., Dippel S., Imvised T., Gueler F., Ure B.M., Kuebler J.F, & Klemann C. (2017). Conventional alpha beta (αβ) T cells do not contribute to acute intestinal ischemia-reperfusion injury in mice. PLoS ONE, 12(7), e0181326.
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7

Immunofluorescent Imaging of Cardiac Tissue

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Immunofluorescence was performed on formalin-fixed paraffin-embedded heart sections (5 µm). Briefly, the sections were deparaffinized, followed by antigen retrieval and blocked with blocking buffer (1% BSA in 1X PBS) for 1 h. Similarly, OCT-embedded sections were fixed with 4% paraformaldehyde for 20 min and rehydrated in PBS for 30 min. Then, sections were incubated overnight in a humidified chamber at 4°C with primary antibody against rat anti-mouse neutrophil (Serotec), rat anti-mouse F4/80 (Serotec), mouse anti-nitrotyrosine (Santa Cruz), mouse anti-4-HNE (Abcam). Finally, sections were incubated with different fluorophore-conjugated secondary antibodies (Invitrogen USA), as described previously (26 (link), 32 (link)).
Zhabyeyev P., Sadasivan C., Shah S., Wang F, & Oudit G.Y. (2023). Amlodipine rescues advanced iron overload cardiomyopathy in hemojuvelin knockout murine model: Clinical implications. Frontiers in Cardiovascular Medicine, 10, 1129349.
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8

Histological Assessment of Kidney Injury

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Tubular injury was assessed using periodic acid-Schiff (PAS)-stained kidney sections. For immunohistochemical staining, we used rat anti-mouse F4/80 (Serotec, Kidlington, UK), Gr-1 (eBioscience, San Diego, CA, USA), CSF-1 (Abcam, Cambridge, UK), TIMP-2 (Abcam), pH3 (Abcam) antibodies. A total of 8–10 high power fields (HPFs) were captured, and the mean number of positive cells was compared between groups.
Kim M.G., Yang J., Ko Y.S., Lee H.Y., Oh S.W., Cho W.Y, & Jo S.K. (2019). Impact of aging on transition of acute kidney injury to chronic kidney disease. Scientific Reports, 9, 18445.
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9

Histopathological Analysis of Tubular Injury

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For (immuno)histological examination, 4μm paraffin sections were cut. Tubular injury was determined on sections stained with periodic acid Schiff after diastase (PasD). The percentage of damaged tubules in the corticomedullary region and in the outer cortex was estimated by a pathologist blinded for the groups using a 5-point scale according to the presence of necrosis in 10 randomly chosen non-overlapping high power fields (hpf, magnification 400x). Injury was graded as follows: 0 = 0%, 1 = <10%, 2 = 10–25%, 3 = 25–50%, 4 = 50–75%, and 5 = >75%. Immunohistochemical stainings to detect macrophages, neutrophils, apoptosis and proliferation were performed using rat-anti-mouse F4/80 (Serotec, Oxford, UK), FITC-labeled anti-mouse Ly-6G (BD Biosciences), rabbit-anti-mouse active caspase 3 (Cell Signaling Technology, Beverly, MA, USA) and rabbit-anti-human Ki67 (clone Sp6; Lab Vision Corp.) respectively. Macrophages and apoptotic and proliferating TEC were counted in the cortico-medullar region in 10 randomly chosen, non-overlapping hpf (magnification 400x). Total number of neutrophils was counted in one kidney sections per mouse.
Stroo I., Claessen N., Teske G.J., Butter L.M., Florquin S, & Leemans J.C. (2015). Deficiency for the Chemokine Monocyte Chemoattractant Protein-1 Aggravates Tubular Damage after Renal Ischemia/Reperfusion Injury. PLoS ONE, 10(4), e0123203.
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10

Macrophage and Neutrophil Depletion in Staphylococcus aureus Infection

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Macrophages were depleted using clodronate liposomes (NvR). The mice were injected i.v. with 1 ml of liposomes per 100 g on day 1 as per manufacturer’s instructions. The mice were then injected with 1x105 CFU of S. aureus NewHG (1:1:1 mixture of NewHG EryR, TetR or KanR) on day 2. Blank liposomes were used as a control. Macrophage depletion was confirmed using histology sections of the liver stained with anti-macrophage antibody (rat anti mouse F4/80, AbD Serotec, catalog number MCA497R). For antibody based neutrophil depletion, in vivo anti-ly6G mouse antibody (1A8, BioXcell, catalog number BE0075-1) was used as per the previously published protocol56 (link). The mice were injected with 1.5 mg/mouse of antibody (200 μl per mouse) on day 1 with the mice being injected with 5x105 CFU S. aureus NewHG (1:1:1 mixture of NewHG EryR, TetR or KanR) on day 2. 100 μl of blood was collected via tail bleeding at the time of S. aureus injection and at the end of the experiment via terminal anaesthesia and heart puncture. The blood samples were mixed with 20 μl of Heparin each and then stained with APC Rat Anti-Mouse Ly-6G antibody (BD biosciences, catalog number 560599) according to the BD bioscience protocol. The samples were then processed using the BD LSRII flow cytometer to confirm neutrophil depletion.
Boldock E., Surewaard B.G., Shamarina D., Na M., Fei Y., Ali A., Williams A., Pollitt E.J., Szkuta P., Morris P., Prajsnar T.K., McCoy K.D., Jin T., Dockrell D.H., van Strijp J.A., Kubes P., Renshaw S.A, & Foster S.J. (2018). Human skin commensals augment Staphylococcus aureus pathogenesis. Nature microbiology, 3(8), 881-890.
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