Evans blue tetrasodium salt

P2 receptor antagonists: Pyridoxal phosphate-6-azo (benzene-2,4-disulfonic acid) tetrasodium salt hydrate (PPADS), MRS2211, MRS2179, MRS2159, A438079, Brilliant Blue G, ATP-2′, 3′-dialdehyde (OxATP), niflumix acid, carbenoxolone disodium salt, and apyrase were purchased from Sigma-Aldrich (St. Louis, MO, United States). NF449, NF023, and Evans Blue tetrasodium salt were purchased from Tocris Bioscience (Abingdon, United Kingdom). Suramin hexasodium salt was purchased from Aladdin (Shanghai, China). Anti-His monoclonal antibody was purchased from EARTHOX Life Sciences (Millbrae, CA, United States). Anti-ETX monoclonal antibody was developed previously by colleagues in our laboratory. HRP-coupled goat anti-mouse IgG antibody was purchased from TransGen Biotech (Beijing, China). The ATP assay kit was purchased from Calbiochem-Merck (Darmstadt, Germany). MTS (3-(4, 5-dimethylthiazol-2-yl) -5(3-carboxymethoxyphenyl) -2-(4-sulfopheny)-2H-tetrazolium, inner salt) was purchased from Promega Corporation (Madison, WI, United States).

Since human IgE is not recognized by murine FcεRI nor FcεRII [22 (link),38 (link),39 (link),40 (link)], BALB/c transgenic mice expressing the α subunit of the human FcεRI and responsible for IgE binding [14 (link),23 (link),25 (link),38 (link)] (a kind gift from Dr. Jean-Pierre Kinet, Beth Israel Deaconess Medical Center, Boston, MA, USA) were used for this assay. Anaphylaxis is a reaction mediated by FcεRI and the assay was performed as previously described with modifications [14 (link),23 (link),25 (link),38 (link)]. Briefly, 0.25 μg of an antibody in 50 μL or vehicle (PBS) control was administered intradermically (i.d.) into the previously shaved skin of transgenic mice. After 1 h, 25 μg of an anti-human κ light chain antibody (Sigma-Aldrich), used to artificially cross-link the human IgE, in 250 μL of 1% Evans blue tetra-sodium salt (Tocris Bioscience, Ellisville, MO, USA) in PBS, was administered intravenously (i.v.) through the tail vein. After 30 min, when visible extravasation of the blue dye into the surrounding tissue was observed, mice were euthanized, and the skin removed. Pictures were analyzed using ImageJ 1.53t (NIH, Bethesda, MD, USA), converting them into 32 bit images, inverted, and displayed as histograms using the histogram command.