Biotin goat anti mouse

For the immunocytochemical analysis, 1x 10⁶ purified eosinophils were incubated in a chamber slide (Nunc Lab-Tek, Sigma-Aldrich, St. Louis, Mo, USA) for 2 hours at 37°C and 5% CO₂ with 10x10⁶L. mexicana promastigotes in 1 ml of RPMI-1640 medium supplemented with 10% heat-inactivated FBS. After incubation, they were washed with PBS and fixed with 2% paraformaldehyde (Sigma-Aldrich, St. Louis, Mo, USA). Nonspecific antigen sites were blocked with 2% bovine serum albumin, dissolved in Tris-HCl, pH 7.6. Thereafter, they were stained with mouse anti-human MBP at a dilution of 1:100 (Santa Cruz Biotechnology, Ca, USA) for 30 min. After washing, eosinophils were incubated with the secondary antibody, biotin goat anti-mouse at a dilution of 1:100 (Invitrogen, Waltham Ma. USA) for 30 min, washed and incubated with streptavidin AP (Alkaline Phosphatase) at a dilution of 1:100 (Thermo Fisher Scientific, Waltham Ma. USA) for 30 min. After washing, they were counterstained with hematoxylin and mounted with resin. Additionally, the incubation of eosinophils with promastigotes was also stained with DAPI in separate samples (Sigma-Aldrich, St. Louis M. USA) to verify the formation of DNA nets.

Samples were loaded on Bolt 4–12% Bis-Tris poliacrylamide gels (Invitrogen), transferred to PVDF and immunoblotted with 4G8 (Signet) diluted 1:1000. The membranes were then sequentially incubated with biotin-goat anti-mouse (Invitrogen) and Streptavidin-horseradish peroxidase conjugate (GE Healthcare) and revealed using ECL Prime (GE Healthcare). The signal intensity was quantified by densitometry using the software Quantity One (BioRad).