*2.7. Histological Staining and Analysis of Lung Tissues*

Lung tissues were fixed in 10% formalin for 10 days and then embedded in paraffin. The sections of lung tissues (4.0–4.5 μm thick) were adhered on Superfrost Plus glass slides (Fisher Scientific, Pittsburgh, PA, USA). After deparaffinization, they were stained with hematoxylin and eosin (H&E) or periodic acid-Schiff (PAS) staining solution. The images were observed through a DP71 digital camera (Olympus, Tokyo, Japan) using a BX51 microscope (Olympus, Tokyo, Japan). A subjective scale from 0 to 3 was used to evaluate the degree of lung inflammation [23]. Briefly, grade 0 indicated no observable inflammation, and grade 1 indicated intermittent cuffing with inflammatory cells. Grade 2 is defined when most vessels or bronchi have slight (1–5 cells thick) layers of inflammatory cells, and grade 3 is defined when most vessels or bronchi have dense (more than 5 cells thick) layers of inflammatory cells. To perform immunofluorescence (IF) staining, the sections of lung tissues (4.0–4.5 μm thick) were precisely adhered on SuperfrostTM Plus slides. After deparaffinization, they were rehydrated and then blocked for 1 h with buffer (PBS containing 1% bovine serum albumin) at RT. Then, the slides were incubated with antibodies against BLT2, MPO, and G-CSF conjugated with Alexa Fluor 488, 594, and 647, respectively, using antibody conjugation kits (Abcam, Cambridge, UK) at 4 ◦C. After washing three times in PBS, they were incubated with 4 ,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich). The slides were processed with washing in PBS and mounted. The images were observed by confocal laser scanning microscopy (LSM 700, Carl Zeiss, Oberkochen, Germany).
