FM 1-43 and FM 1-43X Hair Cell (HC) Staining and Live Imaging

We used FM 1-43 live dye (Invitrogen, #{"type":"entrez-nucleotide","attrs":{"text":"T35356","term_id":"617454","term_text":"T35356"}}T35356) and its fixable analog (FM 1-32FX, Invitrogen, #{"type":"entrez-nucleotide","attrs":{"text":"F35355","term_id":"4820981","term_text":"F35355"}}F35355). For either, we exposed larvae for 30 s (sec) at the desired stage to 3 μM FM 1-43(X), rinsed 3 × 30 s in SW. For the fixable version and at the desired time point post-treatment we anesthetized in ice cold water and subsequently fixed animals (PFA 4%, O/N). Next day we washed larvae 3 × 5 min in SW before mounting them on slides (see above in ICH).

For live imaging, we mounted larvae at the desired post treatment time point using 2% low-melting agarose (LMA) in bottom coverslip chambers. Imaging was performed on an inverted Axiovert (Zeiss) creating z-stacks (in 1 μm steps) from an identified NM in the pLL (L5) using the 63X DIC oil-immersion objective. The first stack was recorded as soon as animals were mounted in LMA, and each following stack was recorded in 30min intervals for a period of 24 h. We recorded the first 4-h immediately post FM 1-43 staining. We used these z-stacks and time-point recordings to re-construct a 24-h post-treatment timeframe.