Prime 95b scmos camera

Widefield imaging was performed on a Leica DMI6000 inverted epifluorescence microscope with excitation/emission filters optimized for DAPI, GFP, Rhodamine, Texas Red and Far-Red fluorophores. Images were acquired with a Photometrics Prime 95B sCMOS Camera (1200 × 1200 11 μm pixels) using the Leica LAS-X acquisition software. Cells were maintained at 37°C in the environmental control chamber (Solent).

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.

Cells grown on glass coverslips were fixed with 4% paraformaldehyde for 10 min and permeabilized with 0.1% Triton-X 100. To prevent non-specific antibody binding, slides were blocked with 1% BSA. Cells were incubated with primary antibody at room temperature for one hour (phospho-histone H2A.X (1:10000, Millipore #05–636); RAD51(1:1000, Abcam); CENPF (1:500, Abcam #5) diluted in 1% BSA. After incubation with secondary antibody (Molecular Probes, Invitrogen, UK) the nuclei were stained 100 ng/µL DAPI. Coverslips were mounted with Mowiol 4–88 (Sigma-Aldrich, UK). Cells were imaged with a Leica DMI6000 inverted epifluorescence microscope with 100x lens and Photometrics Prime 95B sCMOS camera or a Leica SPE single channel confocal laser scanning microscope with 40x lens and Leica DFC365FX monochrome digital camera.