Mouse aortic sinus was serially cut in 7 μm transversal sections, as previously described11 (link),68 (link). Sections from mouse specimens were fixed in acetone and immunostained with specific antibodies anti-mouse CD68 (macrophages, ABD Serotec, Düsseldorf, Germany), anti-mouse Ly-6G (neutrophils, BD PharmingenTM, San Jose, CA, USA), anti-mouse MMP-9 (R&D Systems). Vector Red alkaline phosphatase substrate: SK-5100; in association with Levamisole solution: SP-5000; which produce a magenta coloration is used for revelations (Vector Laboratories, INC, CA, USA). Quantifications were performed using the MetaMorph or Definiens software. Results for other parameters were calculated as percentages of stained area on total lesion area, number of infiltrating cells per mm2 of lesion area or number of lymphatic vessels in adventitia.
Levamisole solution
Manufactured by Vector Laboratories
Sourced in United States
Levamisole solution is a laboratory reagent manufactured by Vector Laboratories. It is a colorless liquid that is used as a phosphatase substrate in various biochemical applications. The solution contains a defined concentration of levamisole, a chemical compound that inhibits the activity of endogenous alkaline phosphatases.
Automatically generated - may contain errors
Lab products found in correlation
Anti mouse cd68, by Bio-Rad (1 mentions)
Anti mouse ly6g, by BD (1 mentions)
Vector red alkaline phosphatase substrate, by Vector Laboratories (1 mentions)
Anti mouse mmp 9, by R&D Systems (1 mentions)
Normal goat serum (ngs), by Agilent Technologies (1 mentions)
Levamisole, by Vector Laboratories (1 mentions)
Goat anti rabbit igg ap sc2007, by Santa Cruz Biotechnology (1 mentions)
Normal goat serum ngs, by Vector Laboratories (1 mentions)
Pe mouse anti human cd14, by BD (1 mentions)
Absorbent pads, by Bio-Rad (1 mentions)
Muse human cd4 t cell kit, by Merck Group (1 mentions)
3 protocols using levamisole solution
1
Immunohistochemical Analysis of Aortic Inflammation
2
Immunohistochemical Staining of CD38 Antigen
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The sections were incubated for 1h in blocking solution, performed with 5% normal goat serum NGS (Vector) in PBS, and incubated overnight, in a humid chamber at 4°C, with primary antibodies that recognize CD38 antigen (goat IgG polyclonal, M-19, sc-7049, Santa Cruz US), diluted 1:100 in normal goat serum (NGS, Dako, Glostrup, Denmark). After several rinses, the samples were incubated for 2 h in secondary antibody (goat anti-rabbit IgG-AP, sc-2007, Santa Cruz), diluted 1:50 in NGS, and washed in Tris-HCl 0.1 M, pH 8.2-8.5 for 5 min. The sections were treated with Levamisole solution (Levamisole, Vector, and 5 mL of Tris-HCl, pH 8.2) for 15 min to inactivate endogenous phosphatase activity, and washed in Tris-HCl 0.1 M, pH 8.2-8.5 for 5 min. The sections were incubated in alkaline phosphatase Kit working solution (Vector) for 15 min in the dark and washed with Tris-HCl pH 8.2 followed by distilled water, 5 min each.
3
Purification and Conjugation of Anti-CD4 Antibody
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Custom purified mouse anti-human CD4 antibody, clone SK3, was bulk ordered from Becton Dickinson (Franklin Lakes, NJ). The purified antibody was custom conjugated with Alkaline Phosphatase (AP) by MOSS Inc. (Pasadena, MD) and supplied at a concentration of 1 mg/ml. Leukoreduction filter LRF10S was purchased from Haemonetics Corporation (Braintree, MA). DynabeadsCD3, DynabeadsCD14, and a magnetic separator rack were purchased from Invitrogen/Thermo Fisher Scientific (Waltham, MA).AP substrate (BCIP/NBT) solution and stabilizing diluent for ALP conjugates from MOSS Inc.; Levamisole solution from Vector Laboratories (Burlingame, CA); Muse ® Human CD4 T Cell Kit from EMD Millipore Corporation (Billerica, MA); PE mouse anti-human CD14 from BD Biosciences (San Jose, CA) and absorbent pads were purchased from Bio-Rad Laboratories (Hercules, CA). All other reagents were purchased from Millipore Sigma (St. Louis, MO).
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