Smz18

Brains, midguts, and abdomens containing fat bodies were dissected in PBS and fixed for 15 min in PBS containing 4% paraformaldehyde. After fixation, the samples were washed with PBS containing 0.2% Triton X-100 (PBST) and blocked with 1% BSA in PBST for 30 min. After incubation with primary antibodies overnight at 4°C: p-ERK (1:100, Cell Signaling, 4370), Prospero (1:100, DSHB, MR1A), ILP2 (1:1000, a kind gift from Hugo Stocker), or Pvf1 (1:50, a kind gift from Ben-Zion Shilo). Tissues were washed and then incubated with secondary antibody and DAPI for 1h, washed, and mounted in Vectashield (Vector). C2C12 myoblasts were cultured and differentiated on cover slides. Treated C2C12 myotubes were washed and fixed for 15 min in PBS containing 4% formaldehyde. After fixation, the samples were washed with PBST, blocked with 1% BSA in PBST, and incubated with primary antibody against MHC (1:50, DSHB, MF20) overnight at 4°C. Cells were then incub ated with secondary antibody and DAPI for 1h, washed and mounted in Vectashield (Vector). Treated 3T3-L1 mature adipocytes were incubated with Bodipy 493/503 (1 μg/mL, Life Technologies, D3922) for 20 min, washed, and imaged. Regular microscopy was performed on a Zeiss Axioskop 2motplus or a Nikon SMZ18 and confocal images were obtained using a Leica system.

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.