Immunohistochemistry

• Mice were euthanized after anesthetized with isoflurane, and the cochlea was dissected from the temporal bone at postnatal day (P) 2. 
• A small hole at the cochlea apex was made, and the oval and round windows were opened. Twenty microliters of 4% PFA in PBS was gently injected through the hole, and the cochlea was incubated with 4% PFA at 4°C overnight. 
• The cochlea was rinsed with 1× PBS briefly, incubated in 1:1 mixture of optimal cutting temperature compound (27050, Ted Pella) and 30% sucrose solution in PBS (v/v) at RT for 1 hour, transferred to embedding mold (18646A-1, Polysciences). 
• The specimens were oriented, lying horizontally flat, with the modiolus mostly parallel to the surface, on the bottom of the mold. This orientation led to obtaining “mid-cochlear” sections during cryosectioning. Any air bubbles around the samples were manually removed with a pair of forceps. Samples were frozen on dry ice. 
• The cochlea was sectioned in 14-μm thickness and attached to the slide glass (22-037-246, Thermo Fisher Scientific) and then dried on the slide warmer at 37°C for 20 minutes. 
• If the staining was not started on the same day, the slides were stored at –80°C in a slide container until immunostaining. 
• All tissue slides were then rehydrated with 500 μl 1× PBS for 5 min at RT and then permeabilized with 500 μl of 0.1% Triton X-100 in 1× PBS for 20 min at RT. 
• The slides were then blocked with the 200 μl blocking solution [10% normal goat serum in PBST (0.1% Trition X-100)] for 1 hour and then were incubated with the primary antibodies in 200 μl 50% blocking solution, mixed with PBST (0.1% Trition X-100) (v/v) at 4°C overnight. 
  • The primary antibodies included α-TECTA produced in rabbit (1:200) and α–Col II produced in mouse (1:100; ab150771, Abcam). 
• After washing the sections three times, each time with 500 μl 1× PBS for 5 min at RT, tissues were treated with secondary antibodies, such as Cy3 AffiniPure Donkey α-Rabbit IgG (H + L) (1:500) and Cy5 Goat α-Mouse IgG (H + L) (1:500), and fluorescein isothiocyanate–PSA (20 μg/ml; L0770, Sigma-Aldrich) with Hoechst 33342 (1:20,000) in 200 μl 50% blocking solution, mixed with PBST (0.1% Trition X-100) (v/v) at RT for 1 hour. 
• After washing the sections three times, each time with 500 μl 1× PBS for 5 min at RT, tissues were treated with fluoromount-G and coverslipped (19811,Thermo Fisher Scientific) for imaging. 

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