C2 silaser scanning confocal microscope

P14 C57Bl6/J WT mice were killed with isoflurane, and then footpad tissue from
hindpaws was removed, fixed overnight in Zamboni’s fixative at 4°C.
Footpads were frozen, and 30 μm sections were prepared. Tissue sections were
blocked in 10% normal goat serum and 0.1% Triton X-100 in PBS for 1 hour
at room temperature and then incubated in primary antibody overnight at 4°C.
Sections were then incubated in secondary antibody (Alexa-Fluor; 1:1000) for 2 hours at
room temperature, counterstained with DAPI (1:1000) and mounted on gelatin-coated slides.
Antibodies used are listed in Supplementary Table 1. Images were acquired using Nikon C2 Silaser-scanning confocal microscope with 60x oil objective.

Live imaging was performed as described (Peters and Berg, 2016 (link)). Briefly, stage 13 egg chambers were individually dissected from flies in room temperature Schneider’s medium. Once isolated, egg chambers were transferred to Schneider’s medium (product number 21720–024, Thermo Fisher Scientific) with LysoTracker (LT, LysoTracker Red DND-99 – Invitrogen by Thermo Fisher Scientific L75283 – 1:1000 dilution) and Hoechst 33342 (product number 62249, Thermo Fisher Scientific– 10 μM). Egg chambers in solution were transferred to the imaging chamber which had a glass bottom. An immobilization blanket (small Kimwipe) was used to keep egg chambers in place during imaging. The immobilization blanket was placed in the solution on top of the egg chambers and a brass washer was placed on the blanket to hold it in place. Live imaging was captured on a Nikon C2+Si laser scanning confocal microscope.