Ab186352

The virus infection on cultured cochlea tissue was confirmed via the presence of mCherry signal. For immunostaining, cochlea tissue was fixed in ice-cold 4% paraformaldehyde, for 10 min and incubated in blocking/permeabilizing buffer (PBS with 5% goat serum and 0.25% Triton-X100). Then, the preparations were incubated overnight at 4°C with rabbit anti-TMEM43 polyclonal (1:100, NBP1-84132, Novus) and mouse anti-TASK-1 (KCNK3) monoclonal (1:100, ab186352, Abcam) diluted in the blocking/permeabilizing buffer. After 3 washes, the cochlear turns were reacted with donkey anti-rabbit Alexa Fluor 594 (1:500, 711-585-152, Jackson ImmunoResearch) donkey anti-mouse Alexa Fluor 488 (1:500, 715-545-150, Jackson ImmunoResearch) in blocking/permeabilizing buffer for 1 h at room temperature. Samples were then rinsed once with blocking/permeabilizing buffer and twice with PBS. Using Fluorsave reagent (345789, Calbiochem), the tissues were mounted on glass slides and covered with a coverslip. Specific immunolabeling was examined under Zeiss confocal microscope. No immunoreactivity was found when the primary antibodies were omitted.

Duolink^® In Situ Red Starter Kit Mouse/Rabbit (DUO92101, Sigma-Aldrich) and far-red detection kit (DUO92013, Sigma-Aldrich) were used. On the experimental day, cochlea tissue (p5~p8) was obtained, fixed with 4% paraformaldehyde, and washed with 0.3% Triton-X100 containing PBS. After blocking, the sample was incubated with rabbit anti-TMEM43 polyclonal (1:100, NBP1-84132, Novus) and mouse anti-TASK-1 (KCNK3) monoclonal (1:100, ab186352, Abcam) at 4℃, overnight. The next day, the sample was incubated with anti-rabbit MINUS and anti-mouse PLUS probe, ligase, and polymerase sequentially. DNA strands participate in rolling circle DNA synthesis only when two probes are in close proximity (<40 nm). Fluorescent-labeled complementary oligonucleotide probes were observed under Zeiss confocal microscopy.