Mvx10 macroview microscope

We imaged adult fish and whole hearts using an MVX-10 MacroView microscope and DP71 camera (Olympus America, Center Valley, PA, USA). We measured fish length (nose to base of fin) using hand-held calipers. We measured ventricle cross-sectional area by tracing the maximal ventricle outline from images of ventricles (atrium removed) positioned in 5% methylcellulose with bulbus arteriosus to the right.

To assess the permeation of clofazimine into normal brain tissue, mice were intraperitoneally injected with 100 mL of a 25 mg/mL suspension of clofazimine in corn oil or vehicle. After 10 minutes of circulation, mice were euthanized, and brains were extracted, snap frozen in isopentane, and sliced into 20 mm sections. Slides were analyzed using a MVX10 MacroView microscope (Olympus) equipped with an ORCA_Flash4.0 v2 sCMOS fluorescent camera (Hamamatsu). A linear range of standards in the brain was developed with varying concentrations (16 μg/mg to 2.5 μg/mg).

For the auxin treatment experiments in Figure S3, fluorescence images were obtained using an Olympus IX83 P2ZF inverted microscope. Z-stacks were captured with a step size of 0.2 μm, using a 100× silicon oil immersion objective (1.35 N.A.). Images in Figures S5 and S6 were taken with an Olympus MVX10 MacroView microscope using a 2× objective (0.5 N.A.). The rest of the DIC images and fluorescence images were obtained using a Nikon Eclipse epifluorescence microscope using 10× (0.25 N.A.) and 40× (0.75 N.A.) objectives. Images were acquired using Olympus cellSens software version 3.1. Before imaging, animals were mounted on 3% agarose pads and immobilized using 10 mM levamisole.

For anatomical analysis, mice were transcardially perfused with phosphate buffered saline (PBS) and then with 4% paraformaldehyde (PFA) in PBS. Brains were extracted from the skulls, post-fixed in 4% PFA overnight at 4°C, and subsequently cut with a vibratome to 80-100 μm thick sequential coronal sections. Slices were collected and mounted in ProLong Gold (Life Technologies) or Vectashield mounting medium containing DAPI (Vector Laboratories H1500). Bright-field and fluorescence images were acquired using an Olympus MVX10 MacroView microscope. For quantification of the cell-specific ablation induced with caspase-3 virus injection in V1 (see sections: Viruses and Viral Injections), cells were counted for every mouse over a distance of 800 μm mediolateral and 400 μm anteroposterior relative to the recording site. The number of cells in ablated areas was compared to the non-ablated right hemisphere from the same animals. For PV cell ablation, cells were counted for every mouse over a distance of 400 μm mediolateral and 400 μm anteroposterior relative to the recording site.

Bright and dark-field images of in situ hybridization were taken with a SPOT insight camera (Diagnostic Instruments, Inc.) operated with SPOT 5.1 software. Live cell images were taken at room temperature using an inverted ECLIPSE TE2000-U Nikon microscope with Image-Pro Plus 7.0 software (Media Cybernetics, Inc.). RT-PCR gels and immunoblot membranes were photographed using a GE Imagequant Luminescent Image Analysis system with accompanying Control Software version 1.2. Live fluorescent and bright field limb organ culture images were obtained at room temperature using an Olympus MVX10 MacroView microscope and Olympus cellSense Standard imaging software version1.3. A Leica TCS LSI confocal microscope with accompanying software was used for imaging of fluorescently labeled frozen tissue sections.