Anti p akt rabbit monoclonal antibodies

Cochlea proteins were incubated in cell lysis buffer (10 mM Tris, pH=7.4, 1% Triton X-100, 150 mM NaCl, 1 mM EDTA, 0.2 mM PMSF) and extracted using a homogenizer. The proteins from the samples (50 μg) were subjected to SDS-polyacrylamide gel electrophoresis and blotted onto a polyvinylidene difluoride membrane. The primary antibodies used were anti-p-Akt rabbit monoclonal antibodies (Millipore; 1 : 200), anti-p27^kip rabbit polyclonal antibodies (Abcam; 1 : 200), and anti-GAPDH mouse monoclonal antibodies (Millipore; 1 : 3333). Subsequently, the membrane was incubated for 1 h at room temperature with the appropriate secondary antibodies coupled with horseradish peroxidase. Finally, the immunoreactive bands were visualized using ECL detection reagents.

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.