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25 protocols using dmrxa2 microscope

1

Subcellular Fluorescence Imaging Protocol

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The subcellular fluorescence experiments were performed by culturing the cells in a 6-well plate (Celltreat) with 10 μM concentration compound solution at 37 °C for 6 h. Afterwards, the medium was removed and cells were washed with 1X PBS three times. Images were acquired using a Leica DMRXA2 microscope with a water immersion objective and DAPI, GFP, and TRITC filter cubes (Chroma Technologies).
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2

Quantifying Cell Migration Dynamics

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For Wound Healing assays, the confluent monolayer was scratched using a pipette tip and incubated with 2.5 μg/ml mitomycin without serum and images were captured using an automated LEICA DMRXA2 microscope at the indicated times. For Boyden chambers assays (Millipore, Molsheim, France), epithelial cells (3 × 105 cells per chamber) were added to the top chamber in low serum (1%)-containing medium. The bottom chamber was filled with low serum (1%)-containing medium. Cells were cultured for 24 h at 37°C. To quantify migration, cells were mechanically removed from the top side of the membrane using a cotton-tipped swab, and migrating cells from the reverse side were fixed with methanol and stained with Giemsa. For each experiment, five representative pictures were taken for each insert, then cells were lysed and absorbance at 560 nm correlated to the amount of Giemsa stain was measured.
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3

Histological Analysis of Pancreatic Tissue

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Frozen sections were prepared by fixing tissues overnight in 4 % paraformaldehyde, followed by cryoprotection in 30 % sucrose at 4 °C for two days and sectioning with given thickness for histological and immunofluorescence analysis. For morphological analysis, Section (5 μm) were stained with hematoxylin and eosin to have images acquired with a Leica DMRXA2 microscope. For immunofluorescence analysis, Section (6–8 μm) were treated following the standard protocol with following antibodies: rabbit anti-OTG1 (Sigma HPA018019, 1:1000), Alexa 488 conjugated rabbit-anti-Giantin (Covance A488-114L, 1:1000), rabbit anti-insulin (Santa Cruz sc-9168, 1:1000), goat anti-glucagon (Santa Cruz sc-7780, 1:1000), donkey anti-goat IgG-FITC (Chemicon AP180F, 1:2000), donkey anti-rabbit IgG-Cy3 (Millipore AP182C, 1:2000).
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4

Histological and Immunoblot Analysis of Mouse Skeletal Muscles

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Muscles and other tissues were frozen in nitrogen-cooled isopentane and liquid nitrogen for histological and immunoblot assays, respectively. Longitudinal and transverse 8 µm cryosections of mouse skeletal muscles were prepared, fixed and stained with antibodies against DHPRα 1 (1:100), RYR1 (1:200), α-actinin (1:1000), DNM2-R2680 (1:200), MTM1-R2827 (1:200), pan-isoform BIN1-C99D (1:50) and BIN1-R2444 (1:100) antibodies. Nuclei were detected by co-staining with Hoechst (Sigma-Aldrich) for 10 min. Samples were viewed using a TCS SP5 laser scanning confocal microscope (Leica). Air-dried transverse sections were fixed and stained with H&E, SDH, NADH-TR or Sirius Red/Fast Green staining and image acquisition performed with a slide scanner NanoZoomer 2 HT equipped with the fluorescence module L11600-21 (Hamamatsu Photonics) or a DMRXA2 microscope (Leica). Cross-sectional area (CSA) was analyzed in H&E sections from TA skeletal muscle using FIJI image analysis software. CSA (μm2) was calculated (>500 fibers per mouse) from 4 to 7 mice per group. The percentage of TA muscle fibers with centralized or internalized nuclei was counted in >500 fibers from 4 to 6 mice using the cell counter plug-in in ImageJ image analysis software.
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5

Kinetics and Effects of SprG Expression

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For the kinetic assays, strains containing the relevant plasmids were grown overnight in BHI. The cultures were then diluted to 1/100 in BHI, then grown in 96-well microtiter plates for 135 min at 37°C until the exponential growth phase (14 (link)). To induce expression of SprG1, SprG2, SprG3 and SprG4, the cultures were then incubated with 1 or 2 μM of aTc. For the kinetics, the optical absorbance at 600 nm (OD600) was measured at 30-min intervals using a Synergy 2 multi-mode reader (Biotek). To determine the effects of SprG induction on bacterial growth, cultures were prepared by 2-fold serial dilutions of exponential phase cultures on BHI plates containing 1 or 2 μM aTc, then incubated for 24 h at 37°C. For the cell death experiments, cultures were incubated for 135 min, washed with 9‰ saline solution, then stained with a LIVE/DEAD bacterial viability kit (Invitrogen). Pictures of fluorescence-labeled cells were captured with a Leica DM RXA2 microscope and a CoolSNAP HQ charge-coupled device camera using Metavue software (Molecular Devices).
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6

Renal Scarring Morphometry Protocol

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The method of morphometry study of renal scarring has been described in previous studies [17, 18] (link). Briefly, Jones’ silver staining was performed on 5 μm thick sections of renal biopsy specimen. Semi-quantitative computerized image analysis was performed with the Leica Twin Pro image analysis system (Leica Microsystems, Wetzlar, Germany), which was connected to a Leica DC500 digital camera on a Leica DMRXA2 microscope with a ×40 objective (final calibration: 0.258 mm/pixel). Image analysis was performed by MetaMorph 4.0 image-analyzing software (Universal Imaging Corporation™, Downingtown, PA, USA). Ten glomeruli and 10 randomly selected areas were assessed in each patient, and the average percentage of scarred glomerular and tubulointerstitial areas, as represented by the area with positive silver staining, were computed.
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7

Imaging Organelle Dynamics in HEp2 Cells

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HEp2 cells were plated in a 6-well plate and allowed to grow overnight. The cells were then exposed to 10 µM of the compound at 37°C for more than 6 hours before adding the organelle tracer (Invitrogen). The working concentrations of organelle tracers were as following: LysoSensor Green 50 nM, MitoTracker Green 250 nM, ER Tracker Blue/white 100 nM, and BODIPY FL C5 Ceramide 50 nM. The organelle tracers were diluted in growing medium and the cells were incubated concurrently with the compound and the tracer for 30 minutes. After adding the tracer for 30 minutes, the loading medium is removed and cells were washed with 1X PBS three times. Images were acquired using a Leica DMRXA2 microscope with a water immersion objective and DAPI, GFP, Texas Red, and TRITC filter cubes (Chroma Technologies).
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8

Live-cell Fluorescent Imaging Microscopy

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A Leica DMRXA2 microscope with 100× objective and a Hamamatsu digital camera interfaced with METAMORPH software (Universal Imaging) was used. Cell morphology and localization of fluorescent protein were visualized in living cells without fixing. Nuclei were stained using mounting medium with DAPI (Vectashield®). Time-lapse microscopy was performed on an inverted confocal laser LSM700 microscope (Carl Zeiss) with an attached temperature chamber and a photometrics coolsnap HQ2 digital camera interfaced with METAMORPH software (Universal Imaging). 1× GMM with 2% agarose or 1× MalMM with 2% agarose was spotted onto glass slides and cooled. Live cells were mounted onto the agar and covered with a cover slip and sealed. Image recoloring and sum projection of z-stacks was performed using Fiji (http://fiji.sc/Fiji).
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9

High-Throughput in situ Hybridization Analysis

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After robotic ISH, slides were cover-slipped and digitally imaged at 1.6 μm/pixel using an automated Leica DM-RXA2 microscope (Carson et al. 2002 (link)). These images were automatically cropped in Adobe Photoshop and stored as TIFF files with LZW lossless compression. TIFFs were uploaded onto the Genepaint.org database where they can be viewed interactively. For Kcnj genes, expression strength and pattern was assigned in 13 major brain regions (cerebral cortex, hippocampus, caudate putamen, globus pallidus, basal forebrain, septum, amygdala, thalamus, hypothalamus, midbrain, pons, ventricles and fiber tracts) using an established atlas-based registration approach (Carson et al. 2004 ). This annotation approach leverages celldetekt software which classifies the spatial area of dye precipitate in each cell resulting from colorimetric detection (Carson et al. 2005a (link)) to annotate using traditional semi-quantitative descriptions of pattern (regional, scattered, ubiquitous) and strength (strong, moderate, weak, none). Pattern assignment is based on the total percentage of cells expressing the mRNA within a region and the scaled weighted deviation in the percentage of cells expressing the gene across the atlas subunits within the structure (Carson et al. 2004 ).
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10

Immunofluorescence Imaging of Human Keratinocytes

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Human keratinocytes were cultured to 70% confluence on glass coverslips and then fixed on ice in either methanol for 2 min or 4% paraformaldehyde for 10 min followed by 0.2% Triton X-100 for 7 min. Primary antibodies described above were detected with Alexa Fluor-conjugated secondary antibodies. Wide field fluorescence microscopy was performed using a DMRXA2 microscope (Leica, Wetzler, Germany) equipped with a 63×/1.32 NA oil immersion objective and narrow band pass filters. Images were acquired with an ORCA digital camera (Hamamatsu Photonics, Bridgewater, NJ) and processed using Simple PCI software (Hamamatsu Corporation, Sewickley, PA). Super-resolution microscopy was performed using a Nikon N-SIM system on an Eclipse Ti-E microscope system equipped with a 100×/1.49 NA oil immersion objective, 488- and 561-nm solid-state lasers in 3D structured illumination microscopy mode. Images were captured using an EM charge-coupled device camera (DU-897, Andor Technology) and reconstructed using NIS-Elements software with the N-SIM module (version 3.22, Nikon). Colocalization analysis was performed by obtaining Mander’s coefficient using ImageJ plugin JACoP [35] (link).
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