Permeabilization buffer

Immunostaining was performed as previously described with minor modifications.^{7 (link),9} Briefly, tissue was fixed by transcardial perfusion of 4% paraformaldehyde (PFA, Alfa Aesar, MA), followed by harvest of the temporal bones and further fixation in 4% PFA at 4°C overnight. After adequate decalcification by incubation in 500mM EDTA (3–6 days), the cochlear ducts were dissected as described by the Eaton-Peabody Laboratories.¹² The tissue was permeabilized with PBS-0.3% Triton X-100 (Sigma-Aldrich, MO) and blocked in permeabilization buffer supplemented with 5% normal goat serum (Cell Signaling Technologies, MA). Pre-synaptic ribbons and post-synaptic densities were labelled using a monoclonal mouse anti-CtBP2 antibody (1:200, BD Biosciences, CA) and a monoclonal mouse anti-GluR2 antibody (1:2000, Sigma-Aldrich, MO), respectively. Following this, the tissue was incubated with the corresponding secondary antibodies, goat anti-mouse IgG2 Alexa Fluor^® 488 and goat anti-mouse IgG1 Alexa Fluor^® 568 (1:1000, ThermoFisher Scientific, MA). Nuclei were counterstained with DAPI. The labelled tissue was mounted with the ProLong Gold antifade reagent (ThermoFisher Scientific, MA).

Macrophages were stimulated for 45 min in the presence or absence of Escherichia coli lipopolysaccharide (LPS) (1 μg/ml) and recovered for analysis. Fc receptors were blocked with normal mouse serum for 15 min at 4 °C. For intracellular detection of NFκB, cells were fixed by incubation in 4% formaldehyde for 10 min at room temperature. Then cells were washed twice with permeabilization buffer (Biolegend). Macrophages were incubated with monoclonal rabbit antimouse pNFκBp65 (Cell signaling) at a 1:50 dilution in permeabilization buffer for 30 min at room temperature. Thereafter, cells were stained with Alexa 647 conjugated goat anti rabbit secondary antibody at a 1:200 dilution at room temperature for 30 min. DAPI was added to the cells for nuclear staining before acquisition.