Bx51 fl

The primary antibodies used were: rabbit anti-XB130 (1:1,000, Abgent, San Diego, CA), rabbit anti-Ki67 (1:100, Lab Vision, Fremont, CA), rat anti-Ly-6B.2 (1:10,000, AbD Serotec, Raleigh, GC), rat anti-F4/80 (1:500, AbD Serotec), goat anti-podoplanin (PDPN) (1:50, R & D Systems, Minneapolis, MN), rabbit anti-surfactant protein C (SFTPC, for type II cells)(1:1,000, Seven Hills Bioreagents, Cincinnati, OH) and rabbit anti-phospho-GSK-3β-Ser9 (1:500, Cell Signaling, Beverly, MA). After incubation with primary antibodies, sections were incubated with appropriate secondary antibodies. IHC was performed using a Vectastain ABC kit (Vector Laboratories, Burlington, Canada) with 3-3-diaminobenzidine as chromogen, and sections were counterstained with hematoxylin, and images were captured via Olympus BX51-FL. For IF, the secondary antibodies used were: donkey anti-goat Alexa Fluor^® 488, donkey anti-rabbit Alexa Fluor^® 555 and goat anti-mouse Alexa Fluor^® 555 (1:200, Invitrogen, Burlington, Canada), and sections were mounted with Prolong Gold Antifade Mountant with DAPI^® (Invitrogen). The slides were examined with an Olympus BX-51, and images were captured via QImaging colour camera (Olympus Co, Ltd). We randomly chose 5 fields (×200) per slide for positive cell counting.

Four groups of animals were used with 5 mice per group: 1) WT-PBS, 2) WT-LPS, 3) Xb130 KO-PBS, 4) Xb130 KO-LPS. At Day 2 after administration, mice were sacrificed, and the lung, spleen, kidney and liver were harvested. The left lung and other tissues was fixed with 4% paraformaldehyde, and the upper lobe of the right lung was used for lung wet/dry (W/D) weight study, whereas the remaining lung tissue was snap-frozen and stored at −80°C. The lung wet/dry (W/D) weight ratio was calculated by dividing the lung tissue weight before and after drying it at 80°C for 48 hours. The lung and other tissues were embedded in paraffin and cut at 5 μm thickness, and the sections were stained with hematoxylin and eosin (H & E). Images were captured using Olympus BX51-FL (Olympus Co, Ltd). We randomly chose 5 fields (×200) and assessed the lung injury with a modified score system [37 (link)]. Briefly, the alveolar edema/exudates, hemorrhage, and interstitial/alveolar cellular infiltration were scored on a scale of 1–3 (0: absent, 1: mild, 2: moderate, 3: severe) with a maximum score of 9 [38 (link)–40 (link)].