Anti rabbit igg alexafluor 594

Immunohistochemical labelling was carried out on fresh tissue from unexposed animals. Mouse cochleae were fixed by scali perfusion of 4% paraformaldehyde, decalcified for 14 days in 8% EDTA and then dissected for surface mount preparation or cryoprotected using sucrose and OCT before being cryosectioned at 50 μm. Nonspecific binding was blocked with 10–15% normal goat or donkey serum, 1% Triton X-100 in phosphate-buffered saline (PBS) for 1 h at room temperature (RT) and sections were then transferred to the primary antibody solution; Neurofilament 200 kDa (Sigma, rabbit, 1:5,000); peripherin (EB12405, Everest Biotech, goat, 1:10,000); CtBP2 (BD Bioscience, mouse, 1:500); vesicular acetylcholine transporter (VAChT; Phoenix, goat, 1:200) in 5–15% normal goat or donkey serum, 0.1% Triton X-100 in PBS) overnight at RT. The sections were washed in PBS and appropriate secondary antibody was applied overnight RT (Molecular Probes, 1:500 anti-rabbit IgG AlexaFluor 594, anti-goat IgG AlexaFluor 488, anti-mouse IgG AlexaFluor 488). Following a further PBS wash, DAPI was applied for 5 min at 1:5,000. Two final washes in PBS were carried out before the tissue was mounted using Vectashield (Vector Laboratories) and imaged on a Zeiss confocal microscope (Zeiss 710 NLO). Data were obtained from 1 to 3 organ of Corti z stacks (30 μm) analysed per animal.