Zen imaging software

Each immunofluorescence experiment was replicated at least twice for identifying spatial expression of genes and 3 times for quantification of in situ signal for differentially expressed genes in the PDGFRα and PDGFRβ-conditional-knockout analysis. For immunofluorescence experiments performed on E12.5 heart sections: Embryos were washed 5 times in PBS after overnight fixation in 4% formaldehyde and stored in 75% ethanol indefinitely until embedding. Embryos were embedded in paraffin processed using standard protocols and embedded for transverse sectioning. Tissue slices were serially sectioned at 5 μm intervals, mounted on slides, and stored at room temperature until the immunofluorescence experiment for paraffin-embedded sections. Sections were imaged with a Zeiss Axio Observer Z1 inverted epifluorescence microscope (Carl Zeiss, Jena, Germany) with Zeiss Axiocam MRm and PCO.edge sCMOS (PCO Imaging) monochrome cameras run by Zeiss Zen imaging software (Carl Zeiss, Jena, Germany). Catalog numbers (Abcam, Cambridge, UK) for Immunofluorescence antibodies used in this study: Anti-PDGFR α: ab234965; Anti-PDGFR beita: ab69506; Anti-α smooth muscle Actin: ab8211; Anti-GM130: ab52649.

Images were processed using Fiji or ZEISS ZEN Imaging Software (Zeiss) and equally adjusted manually if needed. All graphs were generated with Graphpad Prism version 9.0.

Slides were observed by a LSM 880 Zeiss confocal laser microscope (Carl Zeiss S.p.A., Milan, Italy) with the following laser sets: λ = 405–488–543. The height of sections scanning was 1 μm. Eight images per striatum (512 × 512 pixels) were then reconstructed using ZEISS ZEN Imaging Software (Carl Zeiss S.p.A., Milan, Italy) and analyzed with ImageJ (NIH, Bethesda, MA, USA).

After the Dexa scan, axial 200 μm sections were prepared using an EXAKT 300 diamond band saw, ground to a thickness of 80–100 μm with the EXAKT 400CS grinding system and stained with haematoxylin–eosin (HE) without removing the polymethacrylate. Bone morphology surrounding the defect was analysed using ZEISS ZEN Imaging Software (Carl‐Zeiss GmbH, Jena, Germany).

The experimental mice were euthanized at predetermined time points. The mice were anesthetized and perfused with 1.5% paraformaldehyde and 0.1% glutaraldehyde through the left ventricle accessed by a sternotomy. The infrarenal aorta was dissected from surrounding tissue then snap-frozen in optimal cutting temperature (OCT) compound (Sakura, Torrance, CA). Frozen sections were stained with hematoxylin and eosin (H&E) or immunostained with anti-MARCKS antibody (Cat# AB9298; Millipore, Billerica, MA) and anti-Ki-67 antibody (catalog #550609; BD, Franklin Lakes, NJ). The nucleus was counterstained with 4’,6-diamidino-2-phenylindole (DAPI) (Sigma, St. Louis, MO). Confocal microscopic images of the stained sections were acquired using a Zeiss 510 Duo laser confocal microscope (Carl Zeiss, Germany). Zeiss Zen imaging software was used to collect and analyze the image data.

After the cell fixation and permeabilization procedure, the cells were blocked in 5% BSA and subsequently incubated in the anti‐CAV1 (sc‐53564, Santa Cruz) and anti‐NRF2 primary antibodies (CY1851, Abways) overnight at 4°C. On the next day, the cells were washed three times with PBS and incubated in the dark with fluorescence‐labelled secondary antibodies for 1 h. After 3× PBS wash, nuclei staining with DAPI (Sigma‐Aldrich) was conducted for 5 min. For confocal imaging, the fluorescence images were obtained with a confocal laser scanning microscope (Carl Zeiss) and the fluorescence intensity was processed and analysed using ZEISS ZEN Imaging Software (Carl Zeiss).