Third-instar WCS and homozygous spastin5.75 or trans-heterozygous flowerDB25/DB56 larvae were filleted, dissected and immunostained using standard methods (e.g. Ozdowski et al., 2011 (link)). Briefly, larvae were dissected in room temperature PBS and fixed for 30 minutes in 4% paraformaldehyde, immunostained at 4°C overnight using the neuronal membrane marker rabbit anti-HRP (1:100; Jackson ImmunoResearch, PA, 323-005-021) alone or with mAb 22C10 to label microtubules (mouse anti-Futsch, 1:50; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA). Secondary antibodies (Alexa Fluor 488 goat anti-rabbit A-11070 and Alexa Fluor 568 goat anti-mouse A-11031; 1:400; Life Technologies, Grand Island, NY) were incubated for 2–3 hours at room temperature. Fillets were mounted in H-1000 (Vector Laboratories, Burlingame, CA) and z-series images of muscle 4 synapses from larval segments 2–4 acquired on a Zeiss LSM 510 inverted confocal microscope using 63× 1.4 N.A. or 100× 1.2 N.A. PlanApo objectives (Oberkochen, Germany).
Planapo objectives
Manufactured by Zeiss
Sourced in Germany
The PlanApo objectives from Zeiss are high-performance lenses designed for advanced microscopy applications. They provide excellent optical performance, including high numerical aperture, flat field, and chromatic correction, enabling detailed and accurate imaging of samples.
Automatically generated - may contain errors
2 protocols using planapo objectives
1
Larval Neuromuscular Junction Imaging
2
Confocal and TIRFM Imaging Protocols
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Confocal images were acquired with a Laser Scanning Confocal (LSM780; Zeiss) from the PICT-IBiSA @Pasteur Imaging Facility at Institut Curie, equipped with 40× or 100× Plan Apo objectives (numerical apertures, 1.35), and a 1–airy unit pinhole size was used. Single-plane images or z-stack images were acquired. TIRFM was performed using an inverted Nikon microscope Ti-E from the Nikon Imaging Center at Institut Curie-CNRS with a 100× CFI Apo TIRF and equipped with objective (numerical aperture of 1.49), 491 nm and 561 nm lasers, and an EMCCD 512 Evolve camera (Photometrics). Images were analyzed on Fiji (Schindelin et al., 2012 (link)), and ImageJ software and compatible scripts were generated for automated or semiautomated analysis.
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