Leica las x acquisition software

Purified NK cells (8×10⁵ cells and >90% purity) were stained using Mitospy CMX Ros (250 nM, Biolegend) for 30 min at 37°C and fixed in 2% paraformaldehyde (PFA, Sigma) for 15 min at RT, prior to nuclear staining with 4’,6-diamidino-2-phenylindole, dihydrochloride (DAPI, 300 nM, Thermo Fischer Scientific) for 5 min at room temperature. NK cells were mounted using Mowiol (Sigma) and imaged on a Leica SP8 inverted motorized microscope equipped with a ×63/1.4 N.A. oil objective and 405 nm diode and Leica white laser lines. Z-stacks at 0.2 µm increments were captured using an HyD detector in conjunction with Leica LAS X acquisition software.

Frozen brains were sliced into 16 μm (SON) or 20 μm (NTS) coronal sections in a cryostat. A multiplex RNAscope assay was performed using the RNAscope Multiplex Fluorescent Reagent Kit (Advanced Cell Diagnostics, 320850) in accordance with manufacturer's guidelines. RNAscope probes used in this study were purchased from Advanced Cell Diagnostics; Rn-Glp1r-C3 (315221-C3), Rn-Avp-C2 (401421-C2), Rn-Gcg-C2 (315471 C2), Rn-Oxt (479631), and Rn-Fos (403591). Images were captured using a Leica SP5-II confocal laser scanning microscope attached to a DMI 6000 inverted epifluorescence microscope with Leica acquisition software for SON. Images for data analysis were acquired with a Leica SP5-II AOBS confocal laser scanning microscope attached to a Leica DMI 6000 inverted epifluorescence microscope using a 63× PL APO CS lens with a 3.4-zoom factor. Quantification of Glp1r RNA dots in the nucleus (DAPI labelling close to either AVP- or OXT-positive cytoplasm) or cytoplasm (AVP or OXT labelling) of AVP or OXT neurones was performed using a modular workflow plugin for Fiji created by Dr Stephen J Cross from the Wolfson Bioimaging Facility of the University of Bristol, as described [20 ]. All combinations with Gcg were captured using a DMI6000 inverted epifluorescence microscope with Photometrics Prime 95 B sCMOS camera and Leica LAS-X acquisition software.

Organs of Corti were dissected in one contiguous piece at P5 in Leibovitz’s L-15 culture medium (21083–027, Thermo Fisher Scientific) and fixed in 4% formaldehyde for 1 h. The samples were permeabilized with 0.2% Triton-X for 30 min and blocked with 10% goat serum in calcium-free HBSS for 2 h. To visualize the hair cells, samples were labeled with an anti-Myosin 7A antibody (#25–6790 Proteus Biosciences, 1:400) and goat anti-rabbit CF568 (Biotium) secondary antibody. Additionally, samples were labeled with Phalloidin to visualize actin filaments (Biotium CF640R Phalloidin). Samples were then flattened into one turn, mounted on slides using ProLong Diamond Antifade Mounting kit (P36965, Thermo Fisher Scientific), and imaged with a Leica SP8 confocal microscope (Leica Microsystems) using a 63×, 1.3 NA objective. Confocal Z-stacks of 512 × 512 pixel images with an effective pixel size of 288 nm were collected using the tiling functionality of the Leica LASX acquisition software and maximum intensity projected to form 2D images. All experiments were carried out in compliance with ethical regulations and approved by the Animal Care Committee of Massachusetts Eye and Ear.