Cd49b magnetic bead

Peritoneal cells were collected from mice 24 h following CLP. Basophils were then enriched by CD49b⁺ magnetic bead (Miltenyi Biotec) separation. Approximately 2 × 10⁵ CD49b⁺ peritoneal cells were then allowed to adhere to glass slides with cytospin funnels and dried for at least 1 h. The cells were then fixed with methanol followed by incubation with 0.3% H₂O₂ in methanol to inhibit endogenous peroxidase reactions. The cells were then blocked with 5% goat serum and 5% bovine serum albumin in phosphate buffered saline, treated with 5 μg/ml of rat IgG2a anti-mouse Mcpt8 antibody (clone TUG8, catalog number 647401, BioLegend) or with the same amount of an rat isotype control antibody of irrelevant antigen specificity (clone RA3–6B2, catalog number 103201, BioLegend) overnight at 4 °C, and then treated with HRP-conjugated goat anti-rat IgG (catalog number 7077 Cell Signaling Technology) that was diluted 1:500. The cells were subsequently incubated with DAB solution (catalog number D4293, Sigma-Aldrich) and counterstained with May-Grunwald/Giemsa stain. The images were captured with a Leica DM6000 B microscope using a Leica DFC300 FX camera that was run by the Leica LAS X software.