Ab81289

The sections were stained by IHC staining to detect the expression of factors in the VEGF family and α-SMA. After the antigen was repaired, primary antibodies were added at 4°C overnight, and then secondary antibodies were added at room temperature for 1 h, avoiding light. Diaminobenzidine (DAB) was used for staining, and neutral gum was used to seal pieces. The antibodies were anti-VEGFA (1:1000, ab81289, Abcam, United States), anti-VEGFB (1:1000, ab81289, Abcam, United States), anti-VEGFC (1:1000, ab81289, Abcam, United States), and α-SMA (1:1000, 14395-1-AP, Proteintech, United States) (n = 4 each group). The Image J software was used for assessing the mean optical density.

For lung tissues, frozen lung sections were used. For HUVECs, cells cultured on glass coverslips were fixed in 4% paraformaldehyde for 20 min. Frozen lung sections or cells permeabilized in PBS with 0.5% Triton X-100 for 15 min at room temperature. Blocked as above, and then incubated with anti-FGF18 antibody (Santa Cruz, sc-393,471), anti-p65 antibody (Santa Cruz, sc-8008), anti-ICAM-1 antibody (Affinity, DF7413), anti-VCAM-1 antibody (Affinity, DF6082), anti-CD34 (Abcam, ab81289), anti-Aquaporin 5 (Abcam, ab78486), anti-Prosurfactant Protein C (Abcam, ab211326), anti-CD34 (Abcam, ab81289), anti-VE-cadherin (Abcam, ab33168), anti-F4/80 (CST, 30,325 S), anti-CD3 (Proteintech, 17617-1-AP) at 4℃ overnight. And then it was incubated with a secondary antibody of Alexa Fluor 488-conjugated anti-mouse IgG (1:200) (Abcam, ab150113) or Alexa Fluor 647-conjugated anti-rabbit IgG secondary antibody (1:200) (Abcam, ab150075). Finally, the nuclei were stained with DAPI. Images were captured with a Nikon C2si Confocal microscope (Nikon, Japan).

BMMSCs from passage two were suspended in 400 μl of PBS and incubated with each specific antibody. To evaluate surface markers, phycoerythrin (PE)-coupled antibodies against CD29 (25-0291-80, eBioscience, USA), rabbit anti-CD34 (ab81289, Abcam, Cambridge, UK), rabbit anti-CD45 (ab10558, Abcam, Cambridge, UK), mouse anti-CD90 (ab225, Abcam, Cambridge, UK) and mouse anti-CD105 (ab11411, Abcam, Cambridge, UK) were used. The secondary antibodies used in the present study included Cy3-conjugated goat anti-rabbit IgG and goat anti-mouse IgG (Zhuangzhi Co. Ltd, Xi’an, China). After incubation in the dark, the cells were washed with PBS and then resuspended in 400 μl of PBS. Cell fluorescence was determined using a flow cytometer (BD Biosciences, San Jose, CA, USA).
Primary EPCs were suspended in 400 μl of PBS and incubated with each specific antibody. Rabbit anti-CD34 (ab81289, Abcam, Cambridge, UK), rabbit anti-CD133 (ab19898, Abcam, Cambridge, UK) and rabbit anti-VEGFR2 (ab2349, Abcam, Cambridge, UK) were used to identify surface markers. The secondary antibodies used in the present study included Cy3-conjugated goat anti-rabbit IgG (Zhuangzhi Co. Ltd, Xi’an, China). After incubation at 4 °C in the dark, the cells were washed with PBS and then resuspended in 400 μl of PBS. Cell fluorescence was determined using a flow cytometer (BD Biosciences, San Jose, CA, USA).