Mouse/Rat TrkC Antibody

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"Cochleae were stained for NT-3 and BDNF as well as their receptors TrkB, TrkC (both inducing neuroprotective effects on neurons) and p75 (inducing apoptosis in neurons) using immunohistochemistry. Sections were deparaffinized and placed in a microwave for boiling in 10 mM citrate buffer (pH 6) for antigen retrieval. Endogenous peroxidases were blocked by incubation with 0.3% hydrogen peroxide in methanol (MERCK, Hohenbrunn, Germany). Sections were incubated with one of the following primary antibodies: (i) rabbit polyclonal anti-NT3 (1:500 in blocking solution, Alomone Labs Ltd, Jerusalem, Israel); (ii) rabbit polyclonal anti-BDNF (1:500 in blocking solution, Alomone Labs Ltd, Jerusalem, Israel); (iii) rabbit polyclonal anti-TrkB (1:500 in blocking solution, Novus Biologicals Inc., Littleton, CO, USA); (iv) goat monoclonal anti-mouse TrkC (1:200 in blocking solution, R&D Systems, Wiesbaden, Germany); (v) rabbit polyclonal anti-human p75 NTR (1:200 in blocking solution, Alomone Labs Ltd, Jerusalem, Israel) or (vi) blocking solution without primary antibody (negative control) in a humidified chamber overnight. Then, sections were incubated with secondary antibodies (biotinylated anti-rabbit IgG made in goat or anti-goat IgG made in horse according to the primary antibody used; both 1:200, Vector Labs, Burlingame, CA, USA). Binding of the secondary antibody was visualized with streptavidin horse-radish peroxidase (DAKO, Hamburg, Germany) and diaminobenzidine (Vector Labs, Burlingame, CA, USA), which yields a brown reaction product. Between all reaction steps, slides were rinsed with 0.1 M PBS (pH 7.4). Counterstaining was performed using Mayer's hematoxylin.

For assessment of immunohistochemical staining patterns, the most basal spiral ganglion was digitized. Pictures were blinded and printed on 20 × 30 cm color prints. Then, four reviewers (one professor of neuropathology, one assistant professor of neurology, and two residents of neurology with experience in neuropathology) independently grouped the printouts according to (i) homogeneity of the neuronal staining, (ii) intensity of the neuronal staining, and (iii) intensity of extracellular staining. A group was considered to be identified successfully if >80% of blinded slides were grouped together correctly."

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