Filter set 43 he

The protocol for measuring ROS was described previously.^{29 (link),33 (link)} In brief, unfixed cryosections (10 μm) described above were incubated with 5 μM DHE (Invitrogen-Molecular Probes, Eugene, OR, USA) for 20 min at 37 °C. Sections were examined using an Axio Imager (Carl Zeiss) with Filter set 43 HE (Excitation; BP550/25HE, Emission; BP605/70HE, Carl Zeiss). The intensity of staining in the ONL was measured using Image J. All procedures for each sample, from preparing the animals to taking the photographs, were performed at the same time in parallel.

Slides were imaged using a CoolCube 2‐m monochrome camera (#h_0310‐013‐MS; MetaSystems, Newton, MA), Isis Fluorescence Imaging Platform (V5.8.5; MetaSystems, Newton, MA) using both Zeiss EC Plan‐Neofluar 40×/0.75 M27 and Zeiss Plan‐Achromat objectives. Exposure time (never exceeded 120 ms) and upper and lower thresholds were determined using the sample with the highest fluorescence and were consistent between slides of the same experiment each round. Identical sections of the slide were imaged using absolute XY coordinates of the motorized eight‐slide stage which was controlled with a manual movement control system (MMC) (V2.4.5; MetaSystems). DAPI was visualized using 359 nm excitation and 461 nm emission. AF488 was visualized using 495 nm/25 nm excitation and 537 nm/29 nm (wavelength/bandwidth) emission (#49303; Chroma). AF555 was visualized using 550 nm/25 nm excitation and 605 nm/70 nm emission (Filter Set 43 HE; Zeiss). AF647 was visualized using 640 nm/30 nm excitation and 690 nm/50 nm emission (#49009; Chroma).

Seven days after germination, seedlings were immersed in propidium iodide solution (5 μg/ml in water) for 1 min and rinsed three times with water. Root tips were then transferred to slides in a drop of water and covered with a cover slip for observation under the fluorescence microscope with a Zeiss filter set 43HE (adapted from (53 (link))).

Slides were loaded onto a Carl Zeiss AxioImager.Z2 equipped with a PhotoFluor LM-75 halide light source (89 North), a CoolCube 2 m monochrome camera (MetaSystems, #H-0310-013-MS), and a motorized 8 slide stage using a manual movement control system (MMC) (MetaSystems, V2.4.5). Images were captured with the Isis Fluorescence Imaging Platform (MetaSystems, V5.8.5) using a Zeiss EC Plan-Neofluar 40×/0.75 M27 objective and a Zeiss Plan-Achromat 20×/0.8 M27 objective. Slides from the same experiment were imaged using the same settings (exposure time, upper threshold, lower threshold). Nuclear staining with 4′6-Diamidino-2-Phenylindole (DAPI) was visualized using excitation 359 and emission 461 (custom DAPI filter set); AF488 was visualized with excitation 495/25 and emission 537/29 (Chroma, #49303); AF555 was visualized with excitation 550/25 and emission 605/70 (Zeiss, Filter Set 43 HE); AF647 was visualized with excitation 640/30 and emission 690/50 (Chroma, #49009).

Analysis of mitochondrial distribution was performed as previously described14 (link). Cells expressing tagged Cit1p-mCherry from the endogenous locus were imaged on System (1) using a standard rhodamine filter set (Zeiss filter set 43 HE; excitation FT 570, emission 605/70). Images were collected through the entire cell depth (21 z-sections at 0.3-μm intervals), using 1 × 1 binning, 200 ms exposure and analogue gain at 216. Images were deconvolved using an iterative restoration algorithm (Volocity, Perkin-Elmer, Waltham, MA). The relative distribution of mitochondrial mass was estimated by determining total mitochondrial voxel intensity in thresholded, deconvolved wide-field z-series of budding cells, for three equal regions in mothers and two equal regions in daughter cells, as defined in Fig. 1b. Mother and bud compartments were identified in corresponding transmitted-light images. Cells with buds of diameter smaller than 0.2 μm (measured along the mother-bud axis) were excluded.

The AbrNLPs were cloned into the pGWB441 vector to have a C-terminal yellow fluorescent protein (YFP) fusion using Gateway Cloning. Primary cultures of pGWB441-AbrNLP1 and pGWB441-AbrNLP2 grown overnight were used for inoculating secondary cultures, which were grown till they reached an OD₆₀₀ of 0.8–1. The pellets from the secondary cultures were resuspended in the resuspension solution (MES—10 mM, MgCl2—10 mM, and Acetosyringone—200 μM). The resuspended cultures were incubated at room temperature (25°C) for 3–4 h prior to infiltration in 5–6 weeks old N. benthamiana leaves. The subcellular localization of YFP-tagged proteins was examined 2–3 days post infiltration using a Carl Zeiss confocal microscope (LSM880). For visualization of the nucleus and cell wall, the leaves were immersed in 4′,6-diamidino-2-phenylindole (DAPI) solution (10 μg/ml) and Propidium Iodide solution (10 μg/ml) sequentially for 15 min in each solution before imaging. Excitation of the corresponding leaf areas took place at 514 nm for YFP (Filter Set 38 HE, Carl Zeiss AG), at 405 nm for DAPI (Filter Set 49 HE, Carl Zeiss AG), and at 561 nm for PI (Filter Set 43 HE, Carl Zeiss AG).