Af546 goat anti hamster igg

Stimulated neutrophils were collected after 6 hours and stained with Ly6G-FITC antibody (clone 1A8, BioLegend) for 20 minutes. Cells were washed and fixed with 4% PFA (BD Biosciences) overnight. To permeabilize fixed cells, 0.1% TritonX (Fisher Scientific) was used. Cells were incubated with 10% normal donkey serum (NDS, Jackson ImmunoResearch) for 1 hour before addition of primary antibodies: ALF-161 Armenian hamster anti-mouse IL-1α (Fisher Scientific), and rabbit anti-mouse CD63 (clone EPR21151, Ab-cam). Primary antibodies were incubated overnight at 4°C. Cells were washed twice with FACS buffer (PBS with 1% BSA and 2 mM EDTA). AF546 goat anti-hamster IgG (ThermoFisher Scientific) and AF647 donkey anti-rabbit IgG (ThermoFisher Scientific) secondary antibodies were added and incubated at room temperature for 30 minutes. 4 μL of cells were mixed with 4 μL of Vectashield® antifade mounting media with DAPI (Vector Laboratories) and plated on a coverslip. Imaging was done using an LSM700 confocal microscopy (Optical Biology Core, UCI, Leica LSM700) and analyzed with Zen software.

Rat anti-mouse CD45 (clone 30-F11, catalog no. 5505539, 1/400 dilution), CD11b (clone M1/70, catalog no. 550282, 1/400 dilution), and hamster anti-mouse CD11c (clone HL3, catalog no. 550283, 1/50 dilution) were purchased from BD Biosciences (San Jose, CA); F4/80 (clone CI:A3–1, catalogno. MCA497R, 1/400 dilution) and from Bio-Rad Laboratories (Hercules, CA). Goat anti-rat IgG-Alexa Fluor 546 (AF546) (catalog no. A11081, 1/1000 dilution) and goat anti-hamster IgG-AF546 (catalog no. A21111, 1/1000 dilution) were purchased from Thermo Fisher Scientific (Grand Island, NY).