Ar0023

Tumor sections were subjected to deparaffinize and rehydrate with gradient ethanol solutions. After being immersed in antigen retrieval solution (AR0023, Boster, China) and heated for 15 min, sections were then incubated with anti-human Ki-67 antibody (M7240, Dako, Denmark) for 1 h and counterstained with hematoxylin (BA-4226, BASO, China) for 2 min.

Lung tissues of monkeys were fixed, embedded, and cut into slices routinely, followed by deparaffinization in xylene and hydration in gradient ethanol. Slices were then subjected to antigen retrieval in EDTA solutions (Boster, AR0023). And then, samples were incubated with primary antibodies (NR4A3 and CD31 (Abcam, ab28364)) diluted in an antibody diluent (Dako, S-3022) overnight at 4°C. Then, slices were washed three times with PBS and incubated with fluorescence-conjugated secondary antibodies (Invitrogen, A-21202 and A-31572) at room temperature for 1 hour in the dark. After washing three times and staining with Hoechst 33342 (5 μg/mL, Sigma-Aldrich) at room temperature for 5 min in the dark, slices were mounted and imaged by using a Nikon A1 Spectral Confocal Microscope (Nikon, Japan). All images of different groups were captured with the same settings including pinhole size, image resolution, laser power, and gain value, to ensure the comparability of the fluorescent intensity. Measurement of mean fluorescence intensity (MFI) was conducted with NIS-Elements AR (version 4.60.00, 64-bit) software. HE staining of lung tissues was performed routinely following the manufacturer's instructions of the H&E staining kit (Nanjing Jiancheng Bioengineering Institute, D006). Images for HE staining were captured using an inverted Nikon Eclipse Ti-U microscope (Nikon, Japan).