Secondary goat anti rabbit igg h l alexa fluor 488

Inner ears dissected from the heads of mice were fixed overnight in 4% paraformaldehyde at 4°C. Subsequently, the samples were placed in 15% sucrose for 8 h at room temperature for the first dehydration and in 30% sucrose at 4°C overnight for the second dehydration. Finally, the samples were embedded in OCT compound (Sakura Finetek, Torrance, California, USA) and frozen at −20°C until sectioning.
The sections were washed with 10 mM PBS and blocked (10% goat serum) for 1 h at room temperature. Primary antibodies in 5% goat serum were then introduced and the samples were incubated overnight at 4°C. The primary antibodies included anti-myosin VIIa (myo 7a) rabbit polyclonal antibodies (Proteus-Bioscience, Ramona, California, USA; 1 : 200), anti-p27^kip rabbit polyclonal antibody (Abcam, Cambridge, Massachusetts, USA; 1 : 200), and anti-p-Akt rabbit monoclonal antibodies (Millipore, Billerica, Massachusetts, USA; 1 : 200). The samples were washed with PBS and incubated at room temperature for 2 h in secondary goat anti-rabbit IgG (H+L) Alexa Fluor 488 (Invitrogen, Carlsbad, California, USA; 1 : 300) diluted in the same solution as the primary antibodies. A laser scanning confocal microscope (LSM700; Carl Zeiss AG, Pentacon, Germany) was used to analyze the samples.