Goat anti mouse biotinylated secondary antibody

Ten male Long Evans rats (6 young animals, 3–6 months old, and 4 aged animals, 29–33 months old) and 8 Fischer 344 rats (4 young animals, 3 months old, and 4 aged animals, 20–24 months old) were used. The results obtained from the young Long Evans rats have already been published (Ouda et al., 2012b (link)). The tissue was processed as in 2.2.2. coronal serial sections (40 μm thick) were cut with a freezing microtome. Free-floating sections were preincubated in a blocking solution (5% low fat milk in PBS, 1 h) and then immersed in PBS containing the mouse monoclonal antibody SMI-32 against non-phosphorylated heavy and medium neurofilament subunits (1:1000, Covance Research Products, USA) for 18 h (4°C). Sections were incubated with a goat anti-mouse biotinylated secondary antibody (1:200, Sigma-Aldrich) for 1 h, and then with avidin-biotin-peroxidase complex (1:100, Vector Laboratories) for 1 h at room temperature. The reaction was visualized with 0.02% diaminobenzidine (DAB) and 0.01% hydrogen peroxide (15 min). Finally, the sections were mounted on slides, dehydrated and coverslipped.

We performed in situ hybridization using the chick CRABP-I and Fgf10 probes. The chick CRABP-I probe was obtained from the plasmid pBluescript-P, with CRABP-I full-length sequence (413 bp; NM_001030539.2), using HindIII and T7 enzymes to generate the antisense probe. The Fgf10 probe helped to identify the developing sensory patches, as used previously [73 (link)]. All riboprobes were labeled with digoxigenin-11-UTP (Roche, Mannheim, Germany). In situ hybridization was performed according to Ferran et al. [74 ]. To obtain different levels of CRABP-I expression, we used several fixative conditions, different probe concentrations, and distinct incubation times with the chromogenic solution.
Immunohistochemistry was performed in some sections to detect acoustic-vestibular ganglionic neurons using a monoclonal 3A10 antibody (1:40; DSHB, AB_531874; [75 (link),76 (link),77 (link)]. Then, sections were incubated with a goat anti-mouse biotinylated secondary antibody (1:100; Sigma, St. Louis, MO, USA), followed by incubation with ExtrAvidin-biotin-horseradish peroxidase complex (1:200; Sigma). Next, peroxidase activity was revealed by adding a solution containing 0.03% diaminobenzidine (DAB) and 0.005% H₂O₂ to the sections. Finally, sections were mounted with Mowiol mounting solution.