Dmi6000b microscope

Tissues expressing membrane-targeted Cherry or GFP as well as various mRNAs/MOs were dissociated in dissociation buffer, cells were transferred to glass-bottom petri dishes (Fluoro dish, World Precision Instruments) coated with 0.01 mg/ml Fibronectin, and cell behavior was filmed for 2 h. Cells were imaged with a WaveFX spinning disc confocal (Quorum Technologies) mounted on an automated DMI6000B Leica microscope, controlled with Volocity 3DM software (Improvision), using a 40× HCX PL APO CS, NA = 1.25 oil objective. Images were acquired every 2 to 5 min using an EM CCD 512×512 BT camera. Image processing was performed with Metamorph (Universal Imaging Corporation) and Adobe Photoshop7 software. Processing consisted of merging two to three planes from z stacks, assigning pseudo-colors, and adjusting image contrast.

Dissociated cells labelled with GAP-Cherry or GAP-YFP were mixed and plated on glass bottom dish containing 1×MBS-H. Images were acquired using a Quorum technologies WaveFX spinning disc confocal mounted on an automated DMI6000B Leica microscope, with a 40x HCX PL APO CS, NA = 1.25 oil objective was used. Overall, 491 and 561 nm diode lasers were used for GFP and Cherry excitation, respectively. Images were collected with EM CCD 512X512 BT camera using the acquisition software Improvision Volocity 3DM.

Brain tissue was fixed in Carnoy fixative, dehydrated, and embedded in paraffin. Serial sections (7 μm thick) from each block were deparaffinized, hydrated and stained with hematoxylin-eosin (H&E). Then, samples were dehydrated, cleared in xylene and coverslipped with DPX. Samples were visualized with a DMI6000B Leica^® microscope. Brain vacuolation profile was determined on H&E stained sections by scoring the extent of vacuolation in midbrain, hypothalamus, thalamus, hippocampus and motor cortex as previously described24 (link)25 (link).

Carnoy-fixed samples were dehydrated and included in paraffin. 10 μm tissue slices were mounted on glass slides, de-waxed with xylene and hydrated with solutions of decreasing strengths of alcohol (100% to 70%). For PrP^res (PK resistant PrP) staining, slides were rinsed with distilled water and treated with 10 μg/mL PK for 5 min. Samples were subsequently treated with 6% hydrogen peroxide (H₂O₂) for 20 min, rinsed in water, and finally subjected to 3 M guanidinium hydrochloride treatment (prepared in TrisHCl 10 mM, pH 7.8) for 20 min. Slides were incubated overnight using the 6H4 antibody (Prionics, Zurich, Switzerland) and non-specific binding prevented using Dako ARK (Dako, Glostrup, Denmark), following manufacturer’s recommendations. Immunostaining was developed using HRP-conjugated streptavidin visualized with DAB as chromogen. Tissue was later counterstained with haematoxylin for 30 s and rinsed in tap water for 10 min. For Thioflavin S staining, slides were treated with Thioflavin S (1% in ethanol 80%) for 10 min. Samples were washed in distilled water and rinsed quickly (20–30 dips) in 80% alcohol solution. Later, slides were dehydrated in ≥95% ethanol, cleared with xylene, and mounted with resinous mounting medium. Sections were examined under a bright field/epifluorescent DMI6000B Leica microscope (Leica, Buffalo Grove, IL, USA).

For the analysis of early seed development, the oldest closed flower bud of a given inflorescence was emasculated. One day after emasculation, the flowers were pollinated with wild-type pollen and harvested 24 h later.
For whole-mount clearings flowers were vacuum infiltrated in an ethanol:acetic acid solution (9:1) for 30 min, kept at 4°C overnight, washed for 1 h each with 80% and 70% ethanol, and mounted in chloral hydrate:glycerol:water solution (8:2:1; w:v:v). Cytochemical staining of GUS activity was performed on samples as described previously (Vielle-Calzada et al., 2000 (link)). GUS-stained samples as well as cleared whole mounts were then visualized under a Zeiss Axioscope (Zeiss, Oberkochen, Germany) and images were captured by a Canon PowerShot G10 camera. Fluorescence signals were detected by a Leica DMI6000B microscope (Leica Microsystems, Wetzlar, Germany).