Livers containing islet grafts were procured from recipient mice, fixed in 10% formalin neutral buffer solution, embedded in paraffin, and cut into 5 μm-thick sections. Hematoxylin and eosin (H&E) staining was preformed according to standard protocols. Immunohistochemical staining was performed as previously reported.25 (link) Sections were incubated overnight at 4°C with the following primary antibodies: antiinsulin (1:2000; Proteintech, Tokyo, Japan), anti-CD4 (1:100; D7D2Z, Cell Signaling Technology), anti-CD8α (1:400; D4W2Z, Cell Signaling Technology), anti-CD31 (1:100; Cell Signaling Technology), and anti-F4/80 (1:50; Novus Biologicals), followed by incubation for 40 min with a biotinylated secondary antibody diluted 1:300 in PBS. Sections were washed with PBS before addition of avidin-biotin-peroxidase complex (1:100 in biotinylated secondary antibody; ABC-Elite, Vector Laboratories, Burlingame, CA) for 50 min. After washing in PBS, the color reaction was carried out using diaminobenzidine and nuclei were counterstained with hematoxylin. The number of positively stained lymphocytes that had infiltrated into the islet grafts was counted in all lobes of the liver (at the maximum cross section, which contained about 10 islets in total). The areas of liver sections that stained positively for insulin were measured and analyzed using NIH Image J software.
Anti f4 80
Manufactured by Novus Biologicals
Sourced in United States
Anti-F4/80 is a monoclonal antibody that recognizes the F4/80 antigen, a glycoprotein expressed on the surface of mouse macrophages. The F4/80 antigen is a well-established marker for the identification and characterization of mouse macrophages.
Automatically generated - may contain errors
Lab products found in correlation
Anti insulin, by Proteintech (1 mentions)
D7d2z, by Cell Signaling Technology (1 mentions)
D4w2z, by Cell Signaling Technology (1 mentions)
Anti cd31, by Cell Signaling Technology (1 mentions)
Abc elite, by Vector Laboratories (1 mentions)
Laminin, by Agilent Technologies (1 mentions)
Fibronectin, by Abcam (1 mentions)
Anti α sma, by Abcam (1 mentions)
Elastin, by Abcam (1 mentions)
Anti ly6g, by Abcam (1 mentions)
Anti ki67, by Cell Signaling Technology (1 mentions)
Bz x710, by Keyence (1 mentions)
Anti cd31, by Dianova (1 mentions)
Prolong diamond antifade mountant with 4 6 diamidino 2 phenylindole dapi, by Thermo Fisher Scientific (1 mentions)
Anti p erk1 2 thr202 tyr204, by Cell Signaling Technology (1 mentions)
Cell culture reagents, by Merck Group (1 mentions)
Anti β tubulin, by Santa Cruz Biotechnology (1 mentions)
Ib meca, by Merck Group (1 mentions)
Cgs 21680, by Merck Group (1 mentions)
5 protocols using anti f4 80
1
Histological Analysis of Islet Grafts in Liver
2
Comprehensive histological analysis of tumor tissue
3
Immunohistochemical Analysis of Spinal Cord Macrophages
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Mice were sacrificed on day 14 p.i. by transcardial perfusion with saline followed by 4% PF/PBS. The SCs were dissected, post-fixed in 4% PF/PBS, cryoprotected in 27% sucrose/PBS, embedded in optimal cutting temperature (OCT) compound, frozen in a dry ice-ethanol slurry, and stored at − 80 °C. Thirty-micrometer-thick horizontal cryostat sections were obtained from both groups and thaw-mounted on the same slide.
Spinal cord sections were blocked with 10 mM PBS, pH 7.4 containing 10% NGS and 0.5% Triton X-100, for 1 h at RT. Subsequently, they were incubated overnight at 4 °C with anti-F4/80 (Novus Biologicals; 1:500 dilution), anti-Arg1 (MilliporeSigma; 1:500 dilution), and iNOS (BD Biosciences, San Jose, CA, USA; 1:100 dilution) antibodies. Thereafter, the sections were rinsed with PBS and incubated with the corresponding Alexa Fluor 488- or Alexa Fluor 594-tagged secondary antibodies (Thermo Fisher Scientific) for 1 h at RT. Slides were coverslipped with ProLong Diamond antifade mountant with 4′,6-diamidino-2-phenylindole (DAPI; Thermo Fisher Scientific).
Spinal cord sections were blocked with 10 mM PBS, pH 7.4 containing 10% NGS and 0.5% Triton X-100, for 1 h at RT. Subsequently, they were incubated overnight at 4 °C with anti-F4/80 (Novus Biologicals; 1:500 dilution), anti-Arg1 (MilliporeSigma; 1:500 dilution), and iNOS (BD Biosciences, San Jose, CA, USA; 1:100 dilution) antibodies. Thereafter, the sections were rinsed with PBS and incubated with the corresponding Alexa Fluor 488- or Alexa Fluor 594-tagged secondary antibodies (Thermo Fisher Scientific) for 1 h at RT. Slides were coverslipped with ProLong Diamond antifade mountant with 4′,6-diamidino-2-phenylindole (DAPI; Thermo Fisher Scientific).
4
Breast Cancer Cell Phagocytosis Assay
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Breast cancer cells were labelled with 5‐μM carboxyfluorescein succinimidyl ester (CFSE) and incubated for 15 minutes at 37°C in a CO2 incubator protected from light according to the manufacturer's protocol (Invitrogen). Macrophages were incubated in serum‐free medium for 2 hours before adding 2 × 105 CFSE‐labelled breast cancer cells. After coculture at 37°C for 2 hours, cells were harvested, macrophages were stained with APC‐labelled anti‐F4/80 (Novus Biologicals), and phagocytosis were measured and analysed. Phagocytosis was calculated as the percentage of F4/80 + CFSE+ cells among CSFE + cells.
5
Investigating ERK1/2 Signaling in Macrophages
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Monoclonal anti-ERK1/2 and anti-pERK1/2 (Thr202/Tyr204) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA); anti-F4/80 from Novus Biologicals (Littleton, CO, USA); anti-CD31, anti-VEGF-R2, anti-Bad from BD Pharmingen (San Jose, CA, USA); anti-β-tubulin from Santa Cruz Biotechnology (Santa Cruz, CA, USA); recombinant Protein G-HRP conjugate from Thermo Scientific (Waltham, MA, USA). AOPCP (adenosine 5’-alpha,beta-methylene diphosphate), A3AR agonist IB-MECA (N6-(3-Iodobenzyl)adenosine-5′-N-methyluronamide, 1-Deoxy-1-[6-[((3-Iodophenyl)methyl)amino]-9H-purin-9-yl]-N-methyl-β-D-ribofuranuronamide), A2AAR agonist CGS-21680 (2-p-(2-Carboxyethyl)phenethylamino-5′-N-ethylcarboxamidoadenosine hydrochloride hydrate), A1AR agonist CCPA (2-Chloro-N6-cyclopentyladenosine) and cell culture reagents were purchased from Sigma-Aldrich (St Louis, MO, USA).
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