Polyoxyethylene 20 sorbitan monolaurate

The MAP titer was measured by a modified method of Otubo et al. [18 (link)]. Fifty microliters of ethanol-extracted MAP antigens were adsorbed to a 96-well plate (Thermo Fisher Scientific, Inc., MA, USA) by overnight evaporation at room temperature. Three hundred microliters per well of 20% Blocking One^TM (Nacalai Tesque, Inc., Kyoto, Japan) in phosphate buffered saline (PBS, pH 7.2) was added to each well and incubated at 37 °C for 1 h. After rinsing with PBS containing 0.1% Polyoxyethylene(20)Sorbitan Monolaurate (Wako Pure Chemical Industries, Ltd., Osaka, Japan), serum samples were diluted in PBS containing 50 mg/mL of Mycobacterium phlei, which is used to improve specificity in the diagnosis of paratuberculosis. Fifty microliters of a serum sample were added to each well and incubated at 37 °C for 1 h. After rinsing as above, horseradish peroxidase (HRP)-labeled-goat anti-dog IgG antibody, anti-dog IgG₁-HRP or anti-dog IgG₂ (all HRP-labeled antibodies were purchased from Bethyl Laboratories, Inc., Montgomery, Texas, USA, and diluted 1:1000 in PBS) was added to each well and incubated at 37 °C for 1 h. After rinsing again, the substrate (ABTS) was added at 100 µL/well (Roche Diagnostics GmbH, Penzberg, Germany) and absorbance at 415 nm was measured using a microplate reader (Corona Electric Co., Ltd., Ibaraki, Japan).

Serum levels of IgE against Cry j 1 and Cry j 2 were measured using the enzyme-linked immunosorbent assay (ELISA). Purified Cry j 1 or Cry j 2 (Funakoshi Co., Ltd., Tokyo, Japan) was adsorbed onto Immuno 96 microwell^TM plates (Thermo Fisher Scientific Inc., Waltham, MA, USA) by incubating 100 µL in 0.05 M sodium bicarbonate buffer (pH 9.6) in each well at 37 °C for 1 h. After blocking with 300 µL/well 20% Blocking One^TM (Nacalai Tesque, Inc., Kyoto, Japan) in phosphate-buffered saline (PBS, pH 7.2), ELISA plates were incubated at 37 °C for 1 h and rinsed with PBS (pH 7.2) containing 0.1% polyoxyethylene (20) sorbitan monolaurate (Wako Pure Chemical Industries, Ltd., Osaka, Japan). Sera were added at 100 µL/well. Plates were incubated at 37 °C for 1 h and were rinsed as described previously. Horseradish peroxidase conjugated anti-dog IgE antibody was added at 100 µL/well. After incubation for 1 h, plates were rinsed as described previously. Substrate (1% 2, 2-azino-di-(3-ethyl-benzthiazoline sulphonic acid-6) (ABTS)) was added at 100 µL/well (F. Hoffmann-La Roche Ltd., Basel, Switzerland), and absorbance at 415 nm was measured using a microplate reader (Corona Electric Co., Ltd., Ibaraki, Japan).

Mice were sacrificed at day 7 after MI induction. The hearts were minced with fine scissors, and incubated in a cocktail of 0.1% collagenase B (Roche) and 2.2 U/mL Dispase II (Roche) at 37 °C for 30 minutes. Red blood cells were lysed using RBC lysis buffer (eBioscience). The samples were then filtered through a nylon mesh (40μm)39 (link).
Rat serum (Sigma) and Purified Rat Anti-Mouse CD16/CD32 (BD Pharmingen) were added to the cells. Then the cells were incubated with FITC-conjugated Rat anti-Mouse CD45 antibody (BD Pharmingen) or APC-conjugated Rat anti-Mouse CD68 antibody (BioLegend). FITC Rat IgG2bk isotype (BD Pharmingen) or APC-Rat IgG2ak isotype (BioLegend) were used as a control for the anti-CD45 antibody and anti-CD68 antibody, respectively. For the staining with CD 68 antibody the cells were fixed with 0.01% formaldehyde and permeabilized by polyoxyethylene (20) sorbitan monolaurate (Wako) before blocking. Stained cells were diluted with PBS supplemented with 1% FBS before flow analysis of FACS Calibur (BD Biosciences).