M205 fa

Bright-field and fluorescent images were captured using fluorescent dissection
microscopy (Leica M205 FA) or confocal microscopy (Zeiss LSM 700) equipped with
the appropriate filters. Embryos/larvae were anesthetized using
0.16 mg/ml tricaine methanesulfonate (Sigma-Aldrich), then
embedded in 2.5% methyl cellulose (Sigma) or 1% low melting
agarose (BioShop, Burlington, Canada) to obtain the desired orientation. ISH
embryos were balanced between two glass capillary tubes to obtain dorsal views.
The Tg(GBT1300;cmlc2:EGFP) beating heart videos were captured
using Zeiss AxioObserver (Live Cell) (37 ms/frame). Genomic DNA
was extracted from each embryo after imaging and PCR was performed using the
following sets of genotyping primers: (1) gene-specific primers, pdgfra-g-F and
pdgfra-g2-R (intron 16), and (2) gfp-specific primers, gfp2-F
and gfp2-R (Table S1).

Grafted plants were imaged using a Nikon D5300 camera. The CFDA fluorescence was observed with a Leica M205 FA stereofluorescent microscope fitted with a YFP filter. Basic fuchsin-stained samples and β-glucuronidase (GUS)-stained samples were imaged with a Zeiss Axioscope A1 microscope or M205 FA stereofluorescent microscope. The higher-resolution images of hand sections were imaged with an LSM-780 confocal microscope. For CFDA imaging, 488 nm excitation and 500–560 nm emission settings were used. Wavelengths of 561 nm excitation and 571–690 nm emission were used for imaging basic fuchsin-stained samples. All images were analysed using Zen blue or Fiji (version 2.9.0/1.53t)^{59 (link)}.

After perfusion with PBS, tissues were dissected and immediately fixed in 4% paraformaldehyde (PFA). For paraffin sections, samples were dehydrated following standard protocols and sectioned at 7 µm after paraffin embedding for immunofluorescence, hematoxylin/eosin (H&E) and trichrome staining using established techniques. For cryosections, fixed tissues were equilibrated in 30% sucrose/PBS at 4 °C overnight and frozen on dry ice. Sections (7 µm) were mounted on SuperFrost slides for immunofluorescence or periodic acid-Schiff (PAS) staining using a kit from Sigma. Immunofluorescence images were acquired with a Leica M205 FA and a ZEISS Imager Z1. Acquisition of immunohistochemistry and histological images was performed with a ZEISS Axioplan2. 5hmC signals were determined by quantifying the average mean fluorescence intensity (MFI) per nucleus of 100 randomly selected α-SMA⁺ cells in lung tissue section of individual mouse and human subjects using Image J. N numbers refer to the number of individual mouse and human subjects. Antibodies for immunofluorescence staining are listed in Supplementary Table 3.