Lsm 510

Four-day-old seedlings grown in continuous R light (10 µmol m⁻² s⁻¹) were fixed in 2% paraformaldehyde in 1× PBS under vacuum on ice for 15 min and then washed with 50 mM NH₄Cl in 1× PBS for 2 × 5 min, 1× PBS with 0.2% Triton X-100 for 2 × 5 min, and 1× PBS for 3 × 5 min. Fixed seedlings were mounted with ProLong Gold antifade reagent (Thermo Fisher Scientific), sealed with nail polish, and stored at 4 °C until imaging. Nuclei of hypocotyl epidermal cells were imaged using a Zeiss LSM 510 inverted confocal microscope (Zeiss) with a 100×/1.4 Plan-Apochromat oil-immersion objective. YFP was monitored using 514 nm excitation from an argon laser and a 505–550 nm bandpass detector. CFP was monitored using 458 nm excitation from an argon laser and a 470–500 nm bandpass detector. Images were collected using LSM 510 software version 4.2. and processed using Adobe Photoshop CC software (Adobe Systems). The proportion of cells with or without nuclear signals was manually scored. The volume and number of photobodies were analyzed using Huygens Essential software (Scientific Volume Imaging). The object analyzer tool was used to threshold the image and to calculate the volume of photobodies.

10-µm heart cryo-sections were mounted on poly-L-lysine-coated slides and fixed with 4% paraformaldehyde for 20 min, washed with PBS, incubated with rhodamine-linked wheat germ agglutinin (WGA; 1:1000; Vector Laboratories, UK) for 2 h, washed again with PBS and mounted in VectorShield with DAPI (Vector Laboratories, UK). To assess macrophage infiltration, fixed heart cryo-sections were permeabilized with 0.5% Triton X-100 for 20 min, then blocked with 10% horse serum in PBS for 1 h prior to incubation in 10% horse serum/PBS containing 1/100 rat anti-CD68 antibody (Abcam, UK) and 1/1000 WGA for 2 h. The sections were rinsed twice with PBS, incubated with 1/1000 anti-rat IgG-FITC secondary antibody (Abcam, UK) in 10% horse serum/PBS for 1 h, washed three further times with PBS and mounted in VectorShield with DAPI. High-resolution thin-section (0.5 µm) images were taken by confocal microscopy using a Zeiss LSM Axiovert 200M microscope fitted with a 63× water objective, Zeiss LSM510 software and Adobe Photoshop CS. Images in Fig. 1 were taken using a Zeiss AxioImager epifluorescence microscope with AxioVision digital image processing software. CM cross-sectional area counts were averaged from >four animals per group, five fields of view per animal (40× objective) from healthy areas of the left ventricle.