Atg7 rabbit polyclonal antibody

Isolated cochleae were immersed in 4% paraformaldehyde and then decalcified with 0.5 M EDTA (Solarbio, E1170). For cryosectioning, cochleae were immersed in increasing concentrations of 10–30% (w/v) sucrose (Biosharp, Amresco 0335) and then with serial mixtures of OCT (Sakura, Tissue-Tek 4583) and sucrose. The sections and whole mounts were blocked with phosphate-buffered saline blocking solution containing 5% donkey serum, 1% bovine serum albumin, 0.02% sodium azide (Sigma-Aldrich, S8032), and 0.5% Triton; incubated with diluted primary antibodies; and further with fluorescence-conjugated secondary antibodies (Alexa Fluor 488/555/647, Invitrogen). The primary antibodies were Atg7 rabbit polyclonal antibody (Thermo Fisher, PA5-35203, 1:300); Myosin VIIa mouse polyclonal antibody (Thermo Fisher, PA1-936, 1:500); Prestin goat polyclonal antibody (Santa Cruz, sc-22694, 1:200); CtBP2 mouse monoclonal antibody (BD Biosciences, 612044, 1:200); and P62 Guinea Pig polyclonal antibody (Progen, GP62-C, 1:200). The anti-fade Fluoromount-G mounting medium (SouthernBiotech, 0100-01) was used for mounting. The fluorescence images were obtained by a Zeiss LSM 710 confocal microscope. For hematoxylin staining, the whole mounts were stained with diluted hematoxylin (Solarbio, G1080) for 5 min.

Cochleae and other organs were dissected and transferred to the homogenizer tubes and mixed with RIPA lysis buffer (Fudebio, FD008) with a protease inhibitor cocktail (Roche, 11697498001). The supernatant from centrifuged homogenates was mixed with sodium dodecyl sulfate buffer (Beyotime, P0015L), boiled, electrophoresed, and blotted onto a 0.2-μm polyvinylidene difluoride membrane (Millipore, Immobilon ISEQ00010). The primary antibodies were: Atg7 rabbit polyclonal antibody (Thermo Fisher, PA5-35203, 1:500), Prestin goat polyclonal antibody (Santa Cruz, sc-22694, 1:400), GAPDH mouse monoclonal antibody (Abcam, ab9484, 1:1000), β-Actin rabbit IgG antibody (Abmart, P30002, 1:1000), LC3 rabbit polyclonal antibody (CST, 4108, 1:1000), and P62 Guinea Pig polyclonal antibody C-terminal specific (Progen, GP62-C, 1:500). The ECL Kit and horseradish peroxidase-conjugated antibodies were used for detection. Images were obtained by GE ImageQuant LAS4000.