Ab110987

Heads from 5-day-old mice (P5) were bisected, and inner ears plus bone were removed from the skull and then fixed in 4% paraformaldehyde for 2 h at room temperature. Subsequently, specimens were fine-dissected in PBS, then washed and permeabilized in 1% PBS/Triton X-100 (PBT) and blocked with 10% sheep serum. Then, they were incubated with the primary antibody, rabbit polyclonal against Sik3 (ab110987, Abcam, Cambridge, UK, dilution 1 : 200) overnight at 4°C. After washes with PBT, samples were incubated with anti-rabbit Alexa Fluor 488 secondary antibody (Invitrogen, anti-rabbit, diluted 1 : 500) and rhodamine/phalloidin (Invitrogen, diluted 1 : 100). Samples were mounted in Prolong Gold Antifading reagent (Invitrogen). Images were acquired on a LSM 510 Meta confocal microscope (Zeiss, Welwyn Garden City). Image processing was performed in Adobe Photoshop CS5.

The following primary antibodies were used for immunohistochemistry experiments: primary Anti-SIK3 antibody (ab110987, Abcam, Cambridge, UK, and as a control: LS-c120369 SIK3 Lifespan Bioscience, Seattle, USA) at 1 : 50 (P0, P5) or 1 : 40 (4 weeks) concentration; anti-peripherin antibody (ab4666, Abcam, Cambridge, UK) at a 1 : 100 concentration; anti-TUJ1 antibody (Covance, New Jersey, US) at a 1 : 100 concentration; anti-GFAP antibody (ab53554, Abcam, Cambridge, UK) at a 1 : 50 concentration. The secondary antibody, biotin-conjugated donkey anti-rabbit (711-065-152), was purchased from Jackson ImmunoResearch (West Grove, PA, USA). Images of the antibody stained sections were taken using a Zeiss Axioskop MOT light microscope. Image processing was performed in Adobe Photoshop CS5.