Synaptic arbors of muscle 6/7 in the abdominal segment A2 were used for all analyses. Imaging was performed on a Nikon A1R point scanning confocal and a Nikon Yokogawa spinning disc confocal with a Hamamatsu ORCA‐R2 cooled CCD camera. 3D‐SIM was performed on a DeltaVision OMX Blaze microscope (GE Healthcare Life Sciences, Marlborough, MA, USA) with a PCO sCMOS camera. Lasers were adjusted to prevent oversaturation. Images were processed and analyzed with ImageJ and/or MATLAB. Bouton number and size were counted and traced by hand. An HRP mask was used to restrict analysis to neuronal signal for intensity analysis, and MATLAB scripts were used to quantify dTACC and tubulin signals relative to HRP. Line scans were used to create intensity profiles to distinguish different Futsch structures.
Orca r2 cooled
Manufactured by Hamamatsu Photonics
The Orca-R2 is a cooled scientific CMOS camera designed for high-performance imaging applications. It features a large image sensor, high quantum efficiency, and low readout noise, making it suitable for a variety of scientific and industrial imaging tasks.
Automatically generated - may contain errors
Lab products found in correlation
Deltavision omx blaze microscope, by GE Healthcare (1 mentions)
Perfect focus system, by Nikon (1 mentions)
Cyanine3 (cy3), by Thermo Fisher Scientific (1 mentions)
Axiovert microscope, by Hamamatsu Photonics (1 mentions)
Plan apochromat 100 1.4 na oil objective, by Zeiss (1 mentions)
Ix83 microscope, by Olympus (1 mentions)
X100 na 1, by Olympus (1 mentions)
Metamorph software, by Universal Imaging (1 mentions)
4 protocols using orca r2 cooled
1
Imaging and analysis of Drosophila synaptic arbors
2
Live-cell Fluorescence Microscopy
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All images were collected with a wide field Nikon Ti motorized inverted microscope with a Plan Apo 60× NA 1.4 lens objective and the Perfect Focus System for maintenance of focus over time. mNeonGreen fluorescence was excited with a Prior LumenPro fluorescence light source with HQ 480/40, 505lp and ET 535/50 filters (Chroma) for excitation, dichroic and emission, respectively. Images were acquired with a Hamamatsu ORCA-R2 cooled CCD camera controlled with MetaMorph 7.2 software. A 5 × 5 grid of images was obtained with an exposure of 2.5 s (T1) or 0.75 s (T3). Movement of the slide was controlled by Prior Proscan III linear-encoded motorized stage.
3
Immunostaining of Membrane Fractions
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Sonicated membrane fractions were applied on degreased air-dried coverslips and fixed with fixative buffer (2.5% PFA in fractionation buffer) at room temperature for 10 min. Fractions were washed and blocked in blocking buffer for 5 min. Antibodies were placed in blocking buffer and centrifuged briefly at 135,000 rpm. Primary antibodies (including TMR-Star coupled SNAP ligand; New England Biolabs, Ipswich, MA) were incubated on the samples for 15 min, followed by 3× washes for a total of 10 min in fractionation buffer. Secondary antibodies (Alexa Fluor 488 and 647, Jackson Immunoresearch, West Grove, PA; CY3, Molecular Probes) were incubated on the samples for 15 min, followed by the same washing step. Samples were postfixed in fixative buffer for 5 min, washed 1× briefly, and mounted with Mowiol on degreased, air-dried glass slides. Samples were imaged on a Zeiss Axiovert microscope equipped with a Hamamatsu Orca-R2 cooled digital CCD camera using 1 × 1 binning (pixel size, 65 nm), 12-bit pixel depth, and a Zeiss Plan-Apochromat 100×/1.4 NA oil objective.
4
Fluorescence Microscopy of FM4-64
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Fluorescence microscopy was performed using an Olympus IX83 microscope equipped with a x100/NA 1.40 (Olympus) objective and Orca-R2 cooled CCD camera (Hamamatsu), using Metamorph software (Universal Imaging). FM4-64 staining was performed as described previously [32 (link)]. The fluorescence intensities were analyzed by using the program ImageJ V1.44.
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