Mouse anti pou4f3

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.

The intact cochleae were freshly isolated after sacrificing the mice with an overdose of anesthesia and lysed with a lysis buffer containing 2% SDS, 10 mM dithiothreitol, 10% glycerol, a trace amount of Bromophenol Blue and 50 mM Tris HCl, pH 7.4 in 4°C for 1 hour. After homogenization and centrifugation, the protein samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transfer to PVDF membrane. The membrane was respectively probed with mouse anti-Pou4f3 (Santa Cruz Biotechnologies, CA, USA) and mouse anti-GAPDH (BioWorld, MN, USA) antibodies followed by incubation with a corresponding secondary antibody. The signals were visualized by incubation with the ECL substrate (Mucyte, China).