Mab1572

Fish used for immunohistochemistry were fixed for either 2 h at room temperature or overnight at 4°C in 4% paraformaldehyde. Antibody labeling was carried out as previously described (Stawicki et al., 2014 (link)). Fish used for hair cell counts were labeled with either a mouse anti-parvalbumin primary antibody (Millipore, MAB1572) diluted at 1:500 in antibody block (5% heat-inactivated goat serum in PBS, 0.2% Triton, 1% DMSO and 0.2% BSA) or a rabbit anti-parvalbumin primary antibody (Thermo-Fisher Scientific, PA1-933) diluted at 1:1000 in antibody block.

Mice were killed by cervical dislocation at 15 weeks of age and their adrenal glands and brains were collected and stored at −80 °C. The prefrontal cortex and hippocampus were dissected and the hippocampus was separated into ventral and dorsal hippocampus (approximately 50/50). All dissections were performed by the same researcher to ensure consistent dissection ratios. Protein extraction and Western blot analysis were performed as previously described by Klug, Hill [40 (link)] and Buret and van den Buuse [24 (link)]. The primary antibodies were anti-glucocorticoid receptor (GR) (97 kDa, 1:500, ab2768, Abcam), anti-NMDAr2C (1:500, ab110, Abcam) which recognizes 180 kDa GluN2B, 140 kDa GluN2C and 120 kDa GluN1 subunits, anti-NMDAr2A (170 kDa, 1:1000, ab14596, Abcam), anti-parvalbumin (12 kDa, 1:2000, MAB1572, Millipore), and anti-GAD₆₇ (67 kDa, 1:1000, Sigma-Aldrich).

Brains were collected from mice after transcardiac perfusion with sterile PBS and 4% paraformaldehyde (PFA). Brains were post-fixed in 4% PFA for 24 h and incubated in 30% sucrose for an additional 24 h. Fixed brains were frozen and sectioned at 50 µm using a Microm HM550 cryostat. Free-floating sections were washed, then blocked for 1 h at room temperature. Sections were washed, then incubated with primary antibodies (1:1000 dilution): mouse anti-somatostatin (Millipore; MAB354) or rat anti-parvalbumin (Millipore; MAB1572) overnight at 4 °C. Sections were then washed and incubated with conjugated secondary antibody (1:1000 dilution) overnight at 4 °C. Brain sections were mounted on microscope slides and cover-slipped using Fluoromount-G and cell counts were performed by a condition blinded experimenter.

Following exposure to neomycin and perturbagen compound dose responses, zebrafish larvae (n = 9–11 larvae per treatment group, aged 5–7 dpf) were fixed with 4% PFA (in 1X PBS) for 2 h at RT, followed by three 15-min washes in 1X PBS. Zebrafish larvae were then incubated for a 2-h blocking period at room temperature (1% Triton X-100, 5% NGS in PBS). Larvae were then immunostained with mouse anti-parvalbumin primary antibody (monoclonal 1:400, Millipore MAB1572) in primary block (1% Triton X-100, 1% NGS in 1X PBS) at 4°C overnight. Following three 15-min washes in PBS-T (1X PBS, 1% Triton X-100), larvae were transferred into a solution containing a goat anti-mouse secondary antibody conjugated to Alexafluor-488 (1:500) in secondary block (1% NGS in 1X PBS). Larvae were washed in three 15-min washes with PBS-T followed by three 15-min washes in 1X PBS. Larvae were mounted using Fluoromount G on glass slides, and hair cell counts were performed on the SO1, SO2, O1, and OC1 neuromasts using an Axioplan fluorescent microscope (Zeiss) at 40× magnification as previously described (Raible and Kruse, 2000 (link); Harris et al., 2003 (link)).

Fish used for immunohistochemistry were fixed for 2 hr at room temperature in 4% paraformaldehyde. Antibody labeling was carried out as previously described (Stawicki et al. 2014 (link)). Fish used for hair cell counts were labeled with a mouse anti-parvalbumin primary antibody (Millipore, MAB1572) diluted at 1:500 in antibody block (5% heat-inactivated goat serum in PBS, 0.2% Triton, 1% DMSO, and 0.2% BSA). Fish used for the analysis of cilia morphology were labeled with a mouse anti-acetylated tubulin primary antibody (Sigma-Aldrich, 6-11B-1) diluted at 1:1000 in antibody block. For phalloidin labeling, after fixation fish were incubated for 2 hr at room temperature in Alexa Fluor 488 Phalloidin (Molecular Probes) diluted 1:100 in antibody block. They were then washed two times in PBST, one time in PBS, and stored in a 50:50 PBS and glycerol mixture prior to use.