Figure 2.
Analysis of anti-KCC2 antibody specificity. A–F, Anti-KCC2 antibodies directed against the N terminus (anti-nKCC2; A–D) or the C terminus (anti-cKCC2; E, F) were applied to coronal brainstem sections of P12 wild-type (A, C, E) or KCC2 knockdown (B, D, F) mice. A, For anti-nKCC2 in wild-type mice, a moderate IR was observed in sections containing the medial nucleus of the trapezoid body (MNTB) and the LSO, two of the main SOC nuclei. B, Only faint IR was present in KCC2 knockdown mice. C, E, High-magnification confocal images illustrated a similar labeling pattern for anti-nKCC2 and anti-cKCC2 in +/+ mice. D, F, Labeling of the presumed surface of LSO neuron somata (n) and transversely cut dendrites in the neuropil (np) is indicated by arrows and arrowheads, respectively. Both antibodies revealed only faint IR in the LSO of −/− mice. G, No immunoreactive signal was present in the LSO of wild-type mice incubated with secondary antibodies alone. Scale bar: A, B, 200 μm; C–G, 10 μm. H, Immunoblot analysis of anti-nKCC2. Membrane fractions of brains from wild-type (left) or KCC2 knockdown (right) mice were probed with anti-nKCC2. A strong signal in the range of the expected molecular weight was observed in +/+ mice but not in −/− mice. In addition, a weak signal was observed at lower molecular weight, independent of the genotype. 7n, Facial nerve; d, dorsal; l, lateral; Mr, relative molecular mass; +/+, wild-type mice; −/−, KCC2 knockdown mice.