Ab64308

For blotting, gels were transferred to PVDF membranes with 100 V for 60 or 90 min at 4°C for native gels or SDS-PAGE, respectively. Membranes were probed with the following primary antibodies diluted in 5% milk overnight at 4°C: apoA-I antibody (Nr.: ab64308, 1:1,000 dilution; Abcam), apoA-II (Nr.: ab92478, 1:1,000 dilution; Abcam), apoC-I (Nr.: BP2081, 1:1,000 dilution; Acris), serum amyloid A (SAA) (Nr.: RAS-H-SAA-A8, 1:1,000 dilution; courtesy of G. Kostner, Graz, Austria). Membranes were washed and incubated with secondary HRP-conjugated antibodies for 2 h at ambient temperature. Membranes were carefully washed and developed using reagents and detection was performed on a Chemidoc Touch imaging system (Bio-Rad, Vienna, Austria).

Equal amounts (5 μg) of EVs, CEM (T lymphoblast) cell lysate, and two ExoQuick EV-depleted supernatant samples were analyzed using SDS-PAGE followed by immunoblotting with antibodies against known EV protein markers Flotillin1 (clone EPR6041; Abcam), CD9 (EXOAB-CD9A-1; System Biosciences), and the EV purity markers GM130 (ab52649; Abcam) and Apolipoprotein A1 (ab64308; Abcam). Full immunoblots are in Supp. Fig. 2.

Immunohistochemistry was used to examine changes in expression of NF-κB (p65) and Apolipoprotein (Apo) A1 in cochleae. Animals were sacrificed on days 1 or 7 postnoise exposure. Cochleae were fixed with 4% paraformaldehyde at 4°C overnight. After dissection in 0.01 M PBS, the organ of Corti and stria vascularis were collected. Tissues were then permeabilized with 0.25% Triton X-100 in PBS for 30 min, blocked with 5% goat serum in PBS for 30 min, and incubated overnight at 4°C with primary antibody at concentrations recommended by the manufacturer. Tissues were then rinsed with PBS (three times), incubated with secondary antibody at room temperature for 1 h, and counterstained with DAPI for 10 min.
For the noise exposure groups, six cochleae each were used for NF-κB (p65) (6956, Cell Signaling Technology, Inc) and Apo A1 (Abcam, ab64308) staining. Cochleae from six additional animals that were not subjected to noise exposure were used as controls. Several sections of tissue from cochleae were stained only with secondary antibodies to assess nonspecific staining.
Fluorescence was visualized under a confocal microscope (Zeiss LSM780 laser scanning confocal image system) as previously described [24 (link)]. The numbers of different stained hair cells were counted for further quantitative analysis as previously described [25 (link)].