Axioscope epifluorescence microscope

Plants were inoculated with 10⁶ cfu/mL Pst pDSK‐GFPuv (Wang et al., 2007). At 48 h, leaves were cut at the petiole and sections of the lower epidermis were removed using tape and mounted on a glass slide with the epidermis‐less surface facing upwards. Different leaves were stained to detect eDNA or extracellular polysaccharides. A UV filter (435–485 nm) was used during florescence microscopy with an Axioscope epifluorescence microscope (Zeiss) fitted with an AxioCam ICm 1 monochrome camera (Zeiss) and a C‐Mount lens. See File S1 for more details.

For anti-Etv4 and anti-Etv5 staining, thin frozen sections were processed for immunofluorescence as previously described [80 (link)]. Rabbit anti-Etv4 primary antibody, a gift from Dr. Thomas Jessell, was diluted 1:50 in TSP (0.1% Triton X-100, 0.05% [w/v] Saponin in PBS). Rabbit anti-Etv5 (Proteintech 13011-1-AP) was diluted 1:100 in TSP. Fluorescently conjugated secondary antibodies (Jackson ImmunoResearch) were diluted 1:400 in TSP. Samples were imaged on a Zeiss Axioscope epifluorescence microscope.
For anti-calbindin staining, fetal kidneys were fixed overnight at 4˚C in 4% paraformaldehyde in PBS. After washing 3x in PBS, kidneys were mounted in 3% agarose, vibratome-sectioned at a thickness of 50–60 μm, and sections were postfixed in methanol for 15 min. Vibratome sections were blocked with 2% donkey serum in PBT, and primary and secondary antibody incubations were performed in PBT. Sections were incubated with goat anti-calbindin (1:400, Santa Cruz) and rabbit anti-GFP (1:500, Invitrogen) overnight at 4˚C. Following at least three washes with PBS, Cy5-conjugated donkey anti-goat and Cy2-conjugated donkey anti-rabbit secondary antibodies (1: 500) were applied overnight at 4˚C (Jackson ImmunoResearch). Sections were rinsed several times in PBS and mounted on slides with Fluoro-Gel (Electron Microscopy Sciences).