Lsm780 laser scanning confocal microscope

Mice were euthanized with tribromoethanol (Avertin) and fresh brain tissue was harvested and immediately mounted in OCT for cryosectioning. 16–20 μm tissue sections containing the LC were collected for RNAscope-based fluorescence in situ hybridization (ACDBio). Samples were labeled using the RNAscope Multiplex Fluorescent Reagent Kit v2 (ACDBio), and following the supplied protocol. The following probes were used: Calca-C1 (420361) Dbh-O1-C3 (464621-C3), Gad2-C1 (439371) Pdyn-C1 (318771), Penk-C1 (318761), Slc17a6-C2 (319171-C2), Slc17a7-C2 (416631). Probe signal was developed using Opal dyes (Opal 520 and 570 Reagent Pack, Perkin Elmer) at a dilution of 1:1000 and counterstained with DAPI. Fluorescent images were taken using a Leica LSM780 laser scanning confocal microscope.

Mice were euthanized with tribromoethanol (Avertin) and fresh brain tissue was harvested and immediately mounted in OCT for cryosectioning. 20 μm tissue sections containing the LC were collected for Molecular Instruments-based hybridization chain reaction (HCR) in situ hybridization, Samples were labeled using custom probes (Dbh-B2 and Pdyn-B5), and following the supplied protocol. Following RNA amplification and labeling, slides were washed with PBS (3 × 15min), followed by blocking solution (PBS solution containing 0.05% Triton X-100 and 3% BSA) for 1 hour at room temperature. Slides were then incubated overnight at room temperature with GFP-rb antibody (1:500). The following day, slides were washed with a PBS solution containing 0.1% Tween 20 before application of secondary antibodies (1:500) for 2 hours at room temperature. All sections were counterstained in DAPI prior to mounting (Fluoromount-G; SouthernBiotech). Fluorescent images were taken using a Leica LSM780 laser scanning confocal microscope.