Ab28364

Formalin-fixed tissues were routinely processed into paraffin wax blocks and sections cut for Haemotoxylin and Eosin (H&E) staining and immunohistochemistry. Immunohistochemistry was carried out as described previously (Creedon et al., 2016 (link)). Primary antibodies used were CD31 at 1:800 (Abcam, ab28364), ERG (Dako, IR659, ready to use, 200 μl per slide), p53 at 1:2000 (Leica BioSystems, NCL-L-p53-CM5p), PDGFR-β at 1:100 (CST, 3169S), and VE-cadherin at 1:4000 (Abcam, ab33168).

For the immunofluorescence analysis, the cells were fixed using 10% of neutral buffered formalin (Sigma-Aldrich #HT5011, St. Louis, MO, USA) at room temperature for 15 min. After the fixation, the samples were permeabilized for 15 min with PBS containing 0.2% of Triton X-100 and subsequently blocked for 1 h with 5% of BSA in PBST (PBS + 0.1% Tween 20). Following this, the samples were subjected to an overnight incubation at 4 °C in a humid chamber with a primary antibody specific to CD31 (dilution 1:50, DAKO, #M0823, or 1:50, Abcam #ab28364), diluted in antibody diluent (Dako, S3022). GB3/CD77 (1:50, Biolegend, San Diego, CA, USA, clone 5B5) was used for co-staining. On the following day, the samples underwent three washes (5 min each) with PBST and were then incubated at room temperature in a humid, dark chamber for one hour with the appropriate secondary antibody (Thermo Fisher, Waltham, MA, USA; Goat anti-mouse-Alexa Fluor 647 or goat anti-rabbit-Alexa Fluor 488) and diluted 1:500 in the antibody diluent. Following the secondary antibody incubation, the samples were treated with Hoechst (dilution 1:5000) for 15 min at room temperature and visualized either through a fluorescence microscopy (Olympus IX81 light microscope) or a confocal microscopy (Zeiss LSM 800, Oberkochen, Germany).

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).

The femoral muscle tissue was formalin-fixed, paraffin-embedded, and cut into 5-μm-thick sections using a microtome for histological analyses. Using immunohistochemistry, the sections were labeled with a polyclonal anti-antibody (anti-CD31, Abcam, ab28364; anti-αSMA, Dako, M0851) and visualized using the LSAB kit (Dako, Glostrup, Denmark, K0690), which is an automated immunostaining system based on the Lepto-streptavidin–biotin–peroxidase method. The sections were then stained with the corresponding secondary antibodies (Alexa Fluor 555 or Alexa Fluor 488, Molecular Probes, Eugene, OR, USA). The number of CD31-positive cells was evaluated by averaging five visual fields of five sections using an optical microscope (Keyence, Osaka, Japan). The percentage of double-positive cells was calculated by averaging five visual fields of five sections based on fluorescence staining using a confocal microscope (Olympus, Tokyo, Japan).

The gastrocnemius tissues were formalin fixed and paraffin embedded and cut into 5-μm sections using a microtome for histological analyses. The sections were stained by H&E and periodic acid-Schiff (PAS) staining. The images were examined using an optical microscope (Keyence, Osaka, Japan), and quantitative morphometric analysis was performed for each sample using Metamorph software (Molecular Devices, Sunnyvale, CA, USA). Using immunohistochemistry methods, the sections were labeled with polyclonal anti-antibody (anti-CD31; Abcam, ab28364) and were visualized by the LSAB kit (Dako, Glostrup, Denmark, K0690), which is an automated immunostaining system based on the Lepto-streptavidin-biotin-peroxidase method. Again, using immunohistochemistry, the sections were labeled with antibodies (SMA, Dako, M0851; Ki-67, Abcam, ab16667; myogenin, Abcam, ab1835; MyoD, Abcam, ab16148; PAX7, Abcam, ab199010; IPR, Abcam, ab60706), visualized using the corresponding secondary antibodies (Alexa Fluor 488 or Alexa Fluor 555, Molecular Probes, Eugene, OR, USA) that were counterstained by a Hoechst 33342 solution (Dojindo, Kumamoto, Japan, EJ-091), and assessed using a confocal microscope (Olympus, Tokyo, Japan).