Mouse anti muc5ac

PFA fixed ex vivo lung slices were incubated for 1 hour at room temperature in blocking solution: PBS containing 0.1% triton X-100, 0.1% sodium azide, and 2% bovine albumin (BSA), before incubating overnight a 4°C at 1:100 in blocking solution for all primary antibodies used: mouse anti-Muc5ac (Abcam ab3649), rat anti-Ly-6G (Abcam ab2557), rabbit anti-Zfp36l1/l2 (Cell Signaling Technologies). Ex vivo lung slices were washed 3 × 30 min in PBS+0.5% Triton X-100) before incubating overnight at 4°C overnight with: 1:100 Alexa Fluor 488 goat anti-rabbit (Thermo Scientific - A11008) or anti-mouse (A32723) IgG, Alexa Fluor 568 goat anti-rabbit (A11011) or anti-mouse (A11004) IgG, or Alexa Fluor 647 goat anit-rabbit (A32733) or anti-mouse (A21235) IgG +1:250 Alexa Fluor 488, 568, or 647 Phalloidin (Thermo Scientific–A12379, A12380, A22287, respectively). Slices were washed 3 × 30 min in PBS+0.5% Triton X-100, stained with 1:1000 DAPI in PBS for 20 min, mounted in ProLong Gold (Invitrogen P36930), and imaged on a Nikon Eclipse Ti2 spinning disc confocal microscope with a 20X or ×40 objective.