Plan apo 63 1.4 n a

HFF cells infected with the mCherry-DBPv virus were visualized from 20 to 24 hpi. Data acquisition was performed on a CSU-W1 Yokogawa SDC on an inverted Zeiss microscope Observer Z1 equipped with Plan Neo 20×/0.8 NA, Plan Apo 63×/1.4 N.A. The fluorophore was excited with the 561 nm (20 mW) line diode laser combined with a BrightLine Emission filter of 617/73 nm. Images were acquired with an Andor iXon 5078 controlled with Slide Book 6.17 software. Multiple stage positions were collected using a WK-XYBH-APZ30-AV00FT ASI stage controller and optical sections were collected using a Z-stage ASI Piezo MS-2000 Controller. Environmental conditions were controlled by an Okolab H301-K-Frame incubator placed into a Cell Okolab Observer SD enclosure. The set was adjusted to 37 °C and 5% CO₂. A single stack was composed of 37 frames. Each frame (optical slice) had a dimension of 130 µm × 130 µm in XY and 0.27 µm in Z. The Z step between frames was the same than the optical width (0.27 µm). The whole stack acquisition took 7.4 s and the time between stacks (the first frame of one stack and the first frame of the next stack) was 5 min. In all, 13 stacks (timepoints) were acquired and cell focus was maintained throughout the whole acquisition.

Whole-mount IHC was performed as described previously (30 (link), 68 (link)). The following primary antibodies were used: (a) anti-HCN4 (Abcam, ab66501, 1:200 dilution), a polyclonal antibody raised against rat HCN4, and (b) anti-AC_I (Santa Cruz Biotechnology Inc., sc-365350, 1:100 dilution), a monoclonal antibody raised against mouse AC_I. SAN tissue was washed with PBS (3 × 10 minutes) and then incubated with anti-rat and anti-mouse secondary antibodies (Jackson ImmunoResearch Laboratories, 1:1,000 dilution) for 4 hours at room temperature in the dark. It was then washed in PBS (3 × 10 minutes) and incubated for 2 hours with 20% DMSO diluted in PBS. Coverslips were mounted on the slides with ProLong Diamond Antifade Mountant (Thermo Fisher Scientific). The slides were sequentially imaged using a Zeiss 900 confocal laser-scanning microscope equipped with an Airyscan detector module, a Plan-Apo 63× 1.4 NA oil-immersion objective, and 488/561 lasers. Imaris software (Bitplane) was used to perform 3D reconstructions.