Ab300421

The fresh tissues were collected after euthanasia of MMTV-PyVT mice and embedded with opti-mum cutting temperature compound (O.C.T. Compound), frozen at -80°C, and cut into sections for immunofluorescence on a freezing microtome (Leica). Then, sections were incubated in blocking solution (0.4% Triton X-100 and 10% donkey serum in phosphate buffer saline) for 30 min at room temperature, followed by incubating in blocking solution containing primary antibodies overnight at 4°C in the dark. Primary antibodies used were rabbit anti-LY6C (1:200, ab54223, Abcam), rabbit anti-F4/80 (1:200, ab300421, Abcam), rabbit anti-MPO (1:200, 22225-1-AP, Proteintech), armenian hamster anti-CD11c (1:200, MA11C5, ThermoFisher), mouse anti-TPPP3 (1:200, 15057-1-AP, Proteintech), mouse anti-ISG15 (1:50, sc-166755, Santa cruz), mouse anti-IFIT3 (1:50, sc-393512, Santa cruz), and mouse anti-IL12B (1:50, sc-365389, Santa cruz). Sections were washed in wash buffer three times and then incubated in a blocking solution containing secondary antibodies for two hours at room temperature, followed by incubating in 4’,6-diamidino-2-phenylindole (DAPI) (1:5,000, 10236276001, Roche) for 15 min. The paraffin sections of the lung from MMTV-PyVT mice were stained with H&E and analyzed for pathological changes.

Paraffin-embedded tissue sections (4 μm) were soaked in xylene for three times, then rehydration through an ethanol to water gradient at 100%/100%/95%/ 90%/80%/70%. Then, sections were placed in Citrate Antigen Retrieval Solution (0.1 M, pH 6.0) and heated for 5 min. After cooling to room temperature (RT), sections were blocked with 5% BSA for 1 h, then incubated with the anti-arginase antibody (ab233548, EPR22033-369, abcam, England, 1:1000 dilution) and anti-ASS1 antibody (ab170952, EPR12398, abcam, 1:1000 dilution) at 4 °C overnight, or incubated with the anti-iNOS antibody (ab283655, RM1017, abcam, 1:200 dilution) and anti-F4/80 antibody (ab300421, EPR26545-166, abcam, 1:10,000 dilution) at 37 °C for 1 h. After that, sections were washed five times and then incubated with the appropriate secondary antibody (PV-6001, ZSGB-BIO, Beijing, China) for 20 min at RT. Finally, the tissue sections were counterstained with DAPI (C1005, Beyotime) for nuclei visualization. Images were acquired using a scanister (Tissue Gnostics, Austria). To allow comparison between groups, fluorescence intensity was measured using ImageJ software.

All embryos were fixed overnight in 4% paraformaldehyde, dehydrated through graded alcohols, embedded in paraffin, and sectioned at a thickness of 4 µm. For TRAP staining, sections were incubated with pure water at 37 °C for 2 h, placed in the prepared TRAP staining solution at 37 °C for dark staining for 1 h, and then counterstained with haematoxylin. For von Kossa staining, the sections were dropped with the prepared von Kossa silver staining solution and exposed to bright light for 15 min, followed by Hywave solution for 2 min, and finally counterstained with eosin. A TRAP kit (387A-1KT, Sigma, USA) and von Kossa kit (G3282, Solarbio, China) were used as described in our previous study^{9 (link)}. For immunohistochemistry, sections were subjected to antigen retrieval and then blocked with normal goat serum, followed by incubation overnight at 4 °C with the primary antibodies anti-CD14 (1:200, ab181470, Abcam), anti-F4/80 (1:200, ab300421, Abcam), anti-CSF1R (1:200, ab254357, Abcam), and anti-RANKL (1:200, ab45039, Abcam). Anti-CD31 (1:300, ab28364, Abcam) and goat anti-rabbit IgG H&L (1:200, ab150080, Abcam) were used.

For immunohistochemistry (IHC), the sections were incubated with primary antibodies at 4°C overnight. Then, the reacted sections were incubated with the corresponding secondary antibody. After that, the sections were treated with the newly prepared DAB solution. Here, primary antibodies against F4/80 (Abcam, ab300421, 1:5000 dilution, United States), Ki67 (Abcam, ab15580, 1:1000 dilution, United States) and Lgr5 (Affinity, DF2816, 1:200 dilution, United States) were probed, respectively. Finally, the results were observed under a light microscope. All experiments followed the manufacturer’s instructions (Yang X. et al., 2020 (link); Yang H. H. et al., 2020 (link)).