Plan apo na 0.45 objective

Sections were first imaged on a Nikon SMZ18 stereo microscope with automatic stage using a SHR Plan Apo 1X at a zoom magnification of 4x. Stitching was performed directly after imaging using Nikon NIS-Elements software. Sections containing both tdTomato and hGFP positive cells were subsequently imaged by collecting Z-stacks on a Nikon A1 confocal microscope using a 10x Plan Apo NA 0.45 objective (Nikon) for identification of RABV starter neurons, which we identified by co-expression of hGFP and tdTomato. Additional confirmation of starter neurons was done by imaging these neurons again using a 40x Plan Apo NA 0.95 (Nikon) objective.

Tissue sections were washed in PBS and subsequently blocked overnight at 4°C in blocking buffer (PBS containing 1% Triton X-100 (Sigma-Aldrich) and 5% goat serum (Gibco)). The following day, sections were washed 3x for 1 h in PBS containing 0.5% Triton-X100. Next, sections were incubated with primary antibody in PBS containing 0.5% Triton-X100 and 0.5% goat serum for 4 days at 4°C. Sections were then washed 3× 1 h in PBS containing 0.5% Triton-X100 after which they were incubated with secondary antibodies conjugated to Alexa Fluor-488, 546, and 647 of appropriate species (1:500, ThermoFisher Scientific). After several washes in PBS sections were mounted on glass slides and mounted in Fluoromount-G. Z-stacks were collected by imaging on a Nikon A1 confocal microscope using a 10x Plan Apo NA 0.45 objective (Nikon) to identify neuronal identity for Rabv-traced neurons. Primary antibodies used were mouse anti-HA (1:1000, Anti-HA.11, Biolegend), rat anti-somatostatin (1:100, MAB354, Millipore), guinea pig anti-parvalbumin (1:200, 195004, Synaptic Systems), and rabbit anti-DsRed (1:500, 632496, Takara Bio).