Fisherbrand superfrostplus gold microscope slides

Mice that received an AAVrg to direct the expression of tdTom (n = 4) within the sensory neurons that innervate the aortic arch were anesthetized with pentobarbital (50 mg kg^–1, i.p.) and transcardially perfused with RNase-free-isotonic saline followed by 4% paraformaldehyde. The aortic arch and the carotid sinus were collected and stored in RNase-free saline at 4°C for further processing. The left and right nodose ganglia (LNG and RNG, respectively) were dissected and placed in 4% paraformaldehyde for 5 min before they were stored in 20% RNase-free sucrose at 4°C. Brains were extracted and post-fixed for approximately 4 h after which they were stored in 30% RNase-free sucrose at 4°C.
The intact aortic arch, carotid sinus, LNG, and RNG were mounted (whole-mount) onto Fisherbrand Superfrost Plus Gold Microscope Slides (Thermo Fisher Scientific, Waltham, MA, United States). A laser-scanning confocal microscope (Nikon Instruments Inc., Melville, NY, United States) was used to assess viral expression at the aortic arch and the NDG. Tissue from animals with successful expression of the viral constructs were further processed. The LNG and RNG were processed for RNAscope in situ hybridization, and the hindbrains were processed for immunohistochemistry.

Each NDG was sectioned at 10 μm into 6 serial sections using a Leica CM3050 S cryostat (Leica, Buffalo Grove, IL, United States). Sections were immediately mounted onto Fisherbrand SuperFrost Plus Gold Microscope Slides (Thermo Fisher Scientific, Waltham, MA, United States). After air-drying at room temperature for 1 h, slides were rinsed with PBS, incubated in 4% PFA for 5 min, and then dehydrated for 5 min in 50, 75, and 100% EtOH. Following that, slides are incubated in H₂O₂ for 10 min, rinsed with PBS, and allowed to dry for 10–15 min before being stored at –80°C for further processing. The hindbrain was sectioned at 30 μm into 4 serial coronal sections using a Leica CM3050 S cryostat (Leica, Buffalo Grove, IL, United States). Sections were stored in cryoprotective solution at –20°C, until further processing.