Rat anti ly 6g

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.

Histological and IHC examinations were performed using right mandibles at day 7 (N = 3 each) and day 14 (N = 3 each). In the case of day 14, samples were obtained after micro‐CT analysis. The specimens were decalcified with 10% EDTA, embedded in paraffin, and cut into 5‐μm‐thick serial sagittal sections. The sections were stained with hematoxylin and eosin. For IHC examination, the sections were deparaffinized and rehydrated with graded ethanol. Further digestion for antigen retrieval was performed using 0.1% pepsin in 0.01 N hydrochloric acid at room temperature (RT) for 30 min. Nonspecific binding was blocked by incubation in PBS containing 5% skim milk for 1 hr at RT. The sections were then incubated with primary antibodies at 4°C overnight. After washing with PBS containing 0.5% skim milk, the sections were incubated with the secondary antibodies at RT for 1 hr. The primary antibodies were rat anti‐Ly6G (Abcam, Cambridge, UK) and rat anti‐F4/80 (Abcam). The secondary antibody was Alexa Fluor 555 donkey anti‐rat IgG (Life Technologies, Grand Island, NY, USA), and nuclear staining was performed with Hoechst 33342 (Life Technologies). Histological and IHC images were obtained with a Leica CTR6000 microscope. The fluorescence intensities were quantified with ImageJ software (National Institutes of Health, Bethesda, MD, USA).