Sp5ii

Pri-miR-150 and mCherry-GFP-LC3 were co-transfected into cells for 48 h. Nutrient starvation was induced by treating cells with HBSS for 4 h. Fluorescent images of mCherry-GFP-LC3 were acquired by the Leica SP5 II scanning confocal microscope (Leica, Bannockburn, USA).

For the ex vivo data, IHC staining was performed on 10 μm thick canine retinal cryosections that were incubated overnight at +4°C in the presence of rabbit polyclonal anti-canine CNGA3 antibody (courtesy of A. Komáromy, Michigan State University) at a concentration of 1:750. Primary antibody was visualized with Alexa Fluor 568nm goat anti-rabbit secondary antibody and cell nuclei were stained with DAPI. Slides were mounted (Gelvatol, Sigma-Aldrich) and examined by epifluorescence (Axioplan; Carl Zeiss Meditec) or confocal (Leica SP5-II, Leica) microscopy using previously published methods [31 (link)].

Embryonic and adult (MI and sham-operated) hearts were fixed in 0.2% paraformaldehyde (Merck) overnight at 4°C, dehydrated in a sucrose gradient (4% followed of 15%), embedded in gelatin, and frozen. Tissue cryo-sections (4 μm thick) were blocked with either 4% FBS-1% BSA blocking solution or Vector M.O.M. basic kit (Vector Laboratories), depending on the specific conditions detailed in S2 Table. Tissue sections were incubated with primary antibodies overnight at 4°C, followed by 1-hour incubation with Alexa Fluor-conjugated secondary antibodies (see S2 Table for the antibodies list; Invitrogen). Slides were mounted, and nuclei were counterstained with aqueous mounting medium with DAPI (Vector Laboratories). Representative high-resolution images were acquired for each heart structure (At, GV-AVJ, and Vt) at 40× magnification in a confocal microscope (Leica SP5II, Leica, Germany). Whole-heart acquisitions were obtained using the high-content imaging system (IN Cell Analyzer 2000, GE Healthcare).
Isolated fixed CMs were resuspended in 10% FBS-PBS and spun onto superfrost slides in a cytocentrifuge (ThermoFisher). Cytospins were incubated with primary antibodies overnight at 4°C, followed by 1-hour incubation with Alexa Fluor-conjugated secondary antibodies. Acquired images were edited and quantified using the Image J version 1.51d software (NIH).

Mice were sacrificed with an overdose of anesthesia and then infused with phosphate-buffered solution (PBS). The isolated cochlea was fixed with 4% paraformaldehyde (PFA). For plastic sections, decalcification was performed with 10% (w/v) ethylene diamine tetra-acetic acid (EDTA) for 3 days on a shaker followed by gradient dehydration using ethanol. The dehydrated specimens were penetrated and embedded with MC-Plastic I Kit (MuCyte, China) in 4°C overnight. The embedded blocks were cut and stained by hematoxylin/eosin (H&E).
For immunofluorescence of P10 or adult inner ear, whole mounts of cochleae or utricular sensory epithelia were dissected after decalcification with 10% EDTA overnight. The primary antibodies used were rabbit anti-Myosin VIIa (Myo7a) and mouse anti-Pou4f3 (both from Santa Cruz Biotechnologies, USA). Secondary antibodies were Alexa Fluor 546 or Alexa Flour 488-conjugated goat antibodies (Life Technologies, USA). F-actin was labeled by Alexa Fluor 488- or Rhodamine-conjugated phalloidin (Life Technologies, USA). Samples were examined using a Leica SP5-II (Leica, Germany) or Zeiss LSM880 confocal microscope (Zeiss, Germany). ImageJ software (version 1.46r, NIH, MD) was used for image processing of confocal z-stacks. Percentage of OHC loss was defined as number of OHC missing divided by total OHCs demarcated by F-actin stainings.