Journal: Frontiers in Cellular Neuroscience
Article Title: Macrophages Are Dispensable for Postnatal Pruning of the Cochlear Ribbon Synapses
doi: 10.3389/fncel.2021.736120
Figure Lengend Snippet: Complement receptor CR3 was not required for pruning of cochlear ribbon synapses and auditory function. (A,B) RT-qPCR analyses of (A) C3 or (B) CR3 expression in P1, P7, P14, and P28 cochleae. N = 3, error bars represent mean ± SD. * p < 0.05 and ** p < 0.01 by one-way ANOVA. (C) RT-qPCR analyses of CR3 knockout in the cochlea. N = 3, error bars represent mean ± SD. * p < 0.05 by unpaired Student’s t -test. (D,E) Quantitative analyses of OHC (D) and IHC (E) synaptic ribbons in P21 wildtype and CR3 knockout cochlea. (F) Quantitative analyses of putative IHC ribbon synapses (colocalized GluA2 and CtBP2 signals) in P21 wildtype and CR3 knockout cochlea. N = 4–8, error bars represent mean ± SEM. (G) Representative confocal images of P21 wildtype and CR3 knockout cochlea at 16 kHz. Myo7a, hair cells; GluA2, postsynaptic receptors, and CtBP2, pre-synaptic ribbons. (H–L) ABR tests of P21 wildtype and CR3 knockout mice. (H) ABR thresholds, (I) ABR P1 amplitudes, (J) ABR P1 latencies, (K) ABR P2 latencies, and (L) ABR P1-P2 inter-peak latencies. ABR amplitudes and latencies were analyzed at 80 dB SPL. N = 8 (CR3 KO) or 14 (wildtype), error bars represent mean ± SEM.
Article Snippet: The cry-sections or micro-dissected pieces were blocked in 5% normal horse serum (NHS) with 1% Triton X-100 in PBS for 1 h, followed by incubation in primary antibodies (diluted in blocking buffer) at 4°C for 16 h. The primary antibodies used in this study were anti-myosin VIIa (rabbit anti-Myo7a IgG, 25-6790, Proteus Biosciences, 1:500); anti-C-terminal binding protein 2 (mouse anti-CtBP2 IgG1, 612044, BD Biosciences, 1:200); anti-glutamate receptor 2 (mouse anti-GluA2 IgG2a, MAB397, Millipore, 1:2,000); anti-shank1a (rabbit anti-Shank1a IgG, RA19016, NEURONS, 1:1,000), anti-neurofilament H (chicken anti-NFH IgY, AB5539, Millipore, 1:1,000); anti-Iba1 (rabbit anti-Iba1 IgG, 019-19741, WAKO, 1:2,000).
Techniques: Quantitative RT-PCR, Expressing, Knock-Out
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