Dmrxa2 microscope

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.

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).

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 ).

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).