Ab47844

The expression of CD31 in the lung tissues was detected by immunohistochemistry. The sectioned lung tissues were deparaffinized using xylene and dehydrated by graded concentrations of alcohol; they were then placed in a retrieval solution and incubated for 30 minutes in a microwave at high power. Followed by a cooling process, the slides were washed with PBS three times and incubated sequentially with the primary antibody (ab119339; Abcam, Cambridge, UK) at a dilution of 1:50 and biotin-labeled secondary antibody (ab47844; Abcam) at a dilution of 1:1,000. Finally, the slides were stained with 3,3′-diaminobenzidine, counter-stained with hematoxylin, dehydrated, cleared in xylene and fixed. Images were taken under 200× magnification at five different microscopic fields (X71 [U-RFL-T] fluorescence microscope; Olympus).

Left lung samples (4 µm) were washed in water for 15 min following dewaxing (xylene for 5 min; xylene and ethanol (1:5) for 5 min; xylene and ethanol (1:1) for 5 min) and incubated with 3% H₂O₂ for 10 min all at room temperature. Following the antigen was repaired at 95°C for 20 min and the slices were blocked by 0.1% goat serum (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) at room temperature for 15 min. Sections were subsequently incubated with primary antibodies against Ki67 (ab15580, 1:100), p27^Kip1 (ab7961, 1:100) and cyclin B1 (ab2949, 1:100; all provided by Abcam, Cambridge, MA, USA) at 4°C overnight, followed by incubation with second antibody (1% biotin-labeled goat anti-mouse IgG; ab47844; Abcam) at 37°C for 15 min. This was followed by incubation with horseradish peroxidase-labeled streptomyces ovalbumin at 37°C for 15 min and staining with diaminobenzidine (DAB) for 10–30 min at room temperature. The immunohistochemical results were analyzed using Image-Pro Plus software 6.0 (Media Cybernetics, Inc., Rockville, MD, USA) and semi-quantified to obtain integrated optical density (IOD) values. For each animal, 20 random visual fields of view were observed using an optical microscope (magnification, ×400) and an average IOD value calculated.