Alf 161 armenian hamster anti mouse il 1α

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.