Orca er camera

The BiFC assay using MYC fused to the C-terminal fragment of YFP and MAX fused to the N-terminal fragment of YFP has been described previously^{38 (link)}. Fluorescence intensity was analyzed using an automated Axiovert 200M inverted microscope (Zeiss). Images were captured and processed using a Hamamatsu ORCA-ER camera together with software from Improvision (OpenLab 4.0.1). CMV-CFP was cotransfected as an internal standard and the ratio between YFP and CFP fluorescence intensity was calculated and normalized to the value of the DMSO treated cells.

COS7 cells were prelabeled with mouse anti-HA antibodies (Sigma) (60 min, on ice), washed, and incubated in the absence or presence of 5-HT (300 μm) for 60 min at 37 °C. Cells were fixed in 3% paraformaldehyde (in PBS) and washed twice in 50 mm NH₄Cl (in PBS) and blocked (10% FBS, 0.5% BSA in PBS) for 30 min. Subsequent washes and antibody dilutions were performed in PBS containing 10% FBS and 0.5% BSA. Surface receptors were detected using anti-mouse Alexa Fluor 488 (Molecular Probes). Cells were then permeabilized by the addition of 0.5% Triton X-100 (10 min), and the immunofluorescence protocol was repeated from the NH₄Cl step using anti-mouse Alexa Fluor 568 to detect internalized receptors. Cells were examined using a Hamamatsu ORCA-ER camera mounted on a DM-IRB inverted microscope using Volocity software (PerkinElmer Life Sciences).

Embryos were anesthetized in 0.02% tricain (MS-222) in embryo medium and embedded in 1% low melting point agarose. Imaging was performed on a Quorum Technologies spinning-disk confocal microscope (Quorum WaveX Technology Inc Guelph, On, Canada) mounted on an upright Olympus BX61W1 fluorescence microscope with water-immersion lenses. The setup was fitted with a Hamamatsu ORCA-ER camera and image acquisition was done with the Volocity software (Perkin-Elmer) and analyzed with the ImageJ software (NIH). Stacks were acquired at 1 m thickness and assembled in ImageJ. When necessary, adjacent frames were aligned and stitched together using Photoshop CS 5.1 (Adobe Systems Incorporated) Auto-Blend Layers function. For time-lapse microscopy, images were captured every three minutes for 60 minutes. To quantify the primordium migration, the distance between the tip of the primordium at and the tip of the primordium at minutes was measured in ImageJ after superposition of the images.

Lumbrical muscles were normally dissected from the same foot as the FDB preparations, pinned in Sylgard-lined Petri dishes and immersed in MPS containing 5 μg ml⁻¹ tetramethylrhodamine-isothiocyanate conjugated with α-bungarotoxin (TRITC-α-BTX; Invitrogen, USA) to label postsynaptic acetylcholine receptors (AChRs). The preparations were then placed on a rocking platform (Stuart Scientific, Chelmsford, UK) for 10 min, washed twice with MPS for 10 min, fixed in 4% paraformaldehyde (PFA, Electron Microscopy Sciences, Hatfield, PA, USA) for 15 min then washed twice with MPS for 10 min. Once fixed and stained, the muscles were mounted on slides using Vectashield (Vector Laboratories, Peterborough, UK) and imaged on an Olympus BX50WI upright fluorescence microscope fitted with a Hamamatsu Orca-ER camera, captured and processed using OpenLab (Improvision/Perkin-Elmer, Coventry, UK) and Adobe Photoshop (USA) software running on an Apple Mac G5 computer. Confocal images were obtained using a BioRad Radiance 2000 confocal microscope via a Nikon Eclipse EFN600 upright microscope, captured and processed using Zeiss Lasersharp software. Motor endplates were scored as “Occupied” when the TRITC-α-bungarotoxin-labeled endplate showed any coverage with a YFP-labeled axon. Endplates with no overlying, YFP-positive axonal input were scored as “Vacant” (that is, denervated).

Cell nuclei were randomly selected for imaging and analysis, without regards to their Halo, by identifying the nuclear core of each deproteinized nucleus via their intense DAPI fluorescence. Each nuclear core was centered in the field of view and imaged by taking 40 z-stack single plane 12-bit grayscale widefield images at 0.2 μm intervals. All images were captured with a Zeiss Axioplan 2 microscope equipped with a Zeiss Plan-Apochromat 40x/0,95 Korr ∞/0,13–0,21 objective and a Hammamatsu Orca ER camera using Openlab software version 5.5.1 (PerkinElmer). z-stack images for DAPI, FAM, Rho and Cy5 fluorescent channels were captured for each deproteinized nucleus.