0.5 mm glass beads

For tissue harvest, animals were asphyxiated with CO₂, at P14, 21, and 40, rapidly decapitated, and the cochlea separated from the tympanic bulla and otic capsule. Both cochlea from each animal were combined and mechanically lysed in two volumes of XDP lysis buffer and one volume of 0.5 mm glass beads (Next Advance, Averill Park, NY). Lysates were centrifuged, and the protein, which represents at that stage mainly the AN, was separated and stored at −80℃ until analyses. Protein concentration was determined using the DC™ Protein Assay Kit (Bio-Rad, Hercules, CA), and samples (35 µg) were resolved on 4% to 12% bis-tris NuPage gels (Invitrogen, Grand Island, NY). Membranes were probed for anti-ferritin heavy chain (FTH; 1:1000 dilution) for 16 hr at 4℃ (Cell Signaling Technology #3998, Danvers, MA). Immunoreactive proteins were quantified by densitometry using ImageJ software (NIH, Bethesda, MD). Samples were normalized to β-Actin as a loading control and displayed as a ratio to age-matched IS control tissue. Different tissue samples were used for each diet group for each replicate Western blot comparison.

Mouse proteins were extracted from vaginal tissues using SDS extraction buffer (50 mM Tris pH 7.5, 0.5% SDS, Halt Protease inhibitor (Pierce, Rockford, IL) for 1.5 hours on ice after vortexing with 0.5 mm glass beads (Next Advance, Averill Park, NY) five times with 1 min vortexing and 1 min rests. Protein concentration was measured using a Nanodrop spectrophotometer (Thermo Scientific, Rockford, IL) and BCA assay (Pierce, Rockford, IL). Protein lysates (50 µg) were resolved on a 10% SDS-Page gel. The gel was transferred (wet transfer at 300 mA) to a nitrocellulose membrane and then blocked with 5% non-fat dry milk in 1× PBS. For the detection of CD166 antigen in mouse samples, the primary antibody was applied in blocking buffer overnight at 4°C: Secondary antibody was used incubated at room temperature for 1 hour. Following extensive washing, the membranes were scanned using the LI-COR Odyssey imaging system.
To detect OLFM-4 and β-actin, Vk2 cell extracts were resolved on 4–12% Bis-Tris SDS-PAGE gels and mucin 5B was resolved on 3–8% Tris-acetate SDS-PAGE gels f. Gels were transferred to PDVF membranes and probed with the appropriate primary and secondary antibodies and detected using the LI-COR Odyssey imaging system.