Journal: PLoS ONE

Article Title: Immunohistochemical Characterization of the Chemosensory Pulmonary Neuroepithelial Bodies in the Naked Mole-Rat Reveals a Unique Adaptive Phenotype

doi: 10.1371/journal.pone.0112623

Figure Lengend Snippet: Primary and Secondary Antibody Sources and Working Dilutions.

Article Snippet: Rb anti-K v 3.3 (KCN3) pAb , 1∶800 , Alomone Lab.

Techniques:

Journal: PLoS ONE

Article Title: Immunohistochemical Characterization of the Chemosensory Pulmonary Neuroepithelial Bodies in the Naked Mole-Rat Reveals a Unique Adaptive Phenotype

doi: 10.1371/journal.pone.0112623

Figure Lengend Snippet: Primary and Secondary Antibody Sources and Working Dilutions.

Article Snippet: Rb anti-K v 4.3 (KCN4) pAb , 1∶500 , Alomone Lab.

Techniques:

Journal: PLoS ONE

Article Title: BK Channels Mediate Cholinergic Inhibition of High Frequency Cochlear Hair Cells

doi: 10.1371/journal.pone.0013836

Figure Lengend Snippet: (A) Whole-cell currents recorded from an apical outer hair cell in response to voltage steps (100 ms in duration) from −131 mV to 49 mV in 10 mV increments (with an interpulse holding potential of −81 mV) before (black line) and in the presence (grey line) of bath application of 100 nM IBTX, a specific blocker of BK channels, show little IBTX-sensitive currents. (For clarity only some of the traces are shown.) (B) No differences in voltage-gated currents were observed in response to IBTX application across all test potentials examined in apical outer hair cells. (C) Bar plot comparing the fractional contribution of IBTX- and linopirdine-sensitive currents to apical outer hair cells at 49 mV show that apical outer hair cells express predominantly linopirdine-sensitive KCNQ4 currents and little or no BK currents. Confocal micrograph (1 optical section) of an apical cochlear turn immunostained with a monoclonal antibody against the BK channel (red, D and F) shows immunoreactivity in the region corresponding to the neck of the inner hair cells (indicated with an open arrowhead) but no immunoreactivity in the region associated with the outer hair cells (indicated by the solid arrowheads), immunostained with a polyclonal antibody against prestin (green, E and F). Scale bar equals 10 µm.

Article Snippet: Immunofluorescent staining was performed as described previously using the following antibodies: mouse monoclonal anti-BK (L6/23; 1∶500; provided by Dr. James Trimmer, UC Davis, CA), rabbit polyclonal anti BK (APC021; 1∶500; Alomone Labs, Jerusalem, Israel), rabbit polyclonal anti-SK2 (1∶500; provided by Dr John Adelman, Vollum Institute, University of Oregon Health Sciences), goat polyclonal anti-prestin (1∶300; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit polyclonal anti-synapsin (AB1543P; 1∶600; Chemicon/Millipore, Temecula, CA); and mouse monoclonal anti-Na-KATPase α3 subunit (MA3-915; 1∶600; Affinity Bioreagents, Rockford, IL).

Techniques:

Journal: PLoS ONE

Article Title: BK Channels Mediate Cholinergic Inhibition of High Frequency Cochlear Hair Cells

doi: 10.1371/journal.pone.0013836

Figure Lengend Snippet: (A) Whole-cell currents recorded from a basal outer hair cell in response to voltage steps (100 ms in duration) from −131 mV to 49 mV in 10 mV increments (with an interpulse holding potential of −81 mV) before (black line) and in the presence of either 100 µM linopirdine (red trace) or both 100 µM linopirdine and 100 nM IBTX (blue trace). (For clarity only 2 potentials, −131 mV and 49 mV are shown.) (B) At depolarizing membrane potentials, both linopirdine-sensitive KCNQ4 and IBTX-sensitive currents contribute to the voltage gated currents in basal outer hair cells. (C) Bar plot comparing the percent block of I Max at 49 mV in apical and basal OHCs in the presence of 100 µM linopirdine (black bars) or both 100 µM linopirdine and 100 nM IBTX (grey bars). (D) Confocal micrograph (1 optical section) of a basal cochlear turn immunostained with a monoclonal antibody against the BK channel (red, D and F) shows immunoreactivity in the region corresponding to the neck of the inner hair cells (indicated with an open arrowhead) as well as immunoreactivity in the outer hair cells (indicated by the solid arrowheads), immunostained with a polyclonal antibody against prestin (green, E and F). Scale bar equals 10 µm.

Article Snippet: Immunofluorescent staining was performed as described previously using the following antibodies: mouse monoclonal anti-BK (L6/23; 1∶500; provided by Dr. James Trimmer, UC Davis, CA), rabbit polyclonal anti BK (APC021; 1∶500; Alomone Labs, Jerusalem, Israel), rabbit polyclonal anti-SK2 (1∶500; provided by Dr John Adelman, Vollum Institute, University of Oregon Health Sciences), goat polyclonal anti-prestin (1∶300; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit polyclonal anti-synapsin (AB1543P; 1∶600; Chemicon/Millipore, Temecula, CA); and mouse monoclonal anti-Na-KATPase α3 subunit (MA3-915; 1∶600; Affinity Bioreagents, Rockford, IL).

Techniques: Blocking Assay

Journal: PLoS ONE

Article Title: BK Channels Mediate Cholinergic Inhibition of High Frequency Cochlear Hair Cells

doi: 10.1371/journal.pone.0013836

Figure Lengend Snippet: 3D renderings of confocal z-stacks of apical (A), middle (B), and basal (C) cochlear turns immunostained with a monoclonal antibody against the BK channel (red) and a polyclonal antibody against synapsin (green) to label the efferent presynaptic terminals show that BK channel immunoreactivity in the three rows of outer hair cells (when present) is associated with efferent terminals. (D) Moreover, the size of BK channel immunopuncta increases from apical to middle and basal turns, paralleling an increase in size and likely number of efferent terminals. Data are presented as box plots representing the median (box interior), the 25th and 75th percentile (box boundaries), the 10th and 90th percentile (whiskers), and the 5th and 95th percentile (dots). (E) 3D renderings of confocal z-stacks of a middle cochlear turn immunostained with a polyclonal antibody against the BK channel (red) and a monoclonal antibody against the NaK-ATPase α3 (green) to label the efferent presynaptic terminal membrane shows adjacent but not co localized expression of the BK channel with the presynaptic efferent terminal membrane. The location of the three rows of outer hair cells are indicated (solid arrowheads). Grid dimensions equal 10 µm.

Article Snippet: Immunofluorescent staining was performed as described previously using the following antibodies: mouse monoclonal anti-BK (L6/23; 1∶500; provided by Dr. James Trimmer, UC Davis, CA), rabbit polyclonal anti BK (APC021; 1∶500; Alomone Labs, Jerusalem, Israel), rabbit polyclonal anti-SK2 (1∶500; provided by Dr John Adelman, Vollum Institute, University of Oregon Health Sciences), goat polyclonal anti-prestin (1∶300; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit polyclonal anti-synapsin (AB1543P; 1∶600; Chemicon/Millipore, Temecula, CA); and mouse monoclonal anti-Na-KATPase α3 subunit (MA3-915; 1∶600; Affinity Bioreagents, Rockford, IL).

Techniques: Expressing

Journal: PLoS ONE

Article Title: BK Channels Mediate Cholinergic Inhibition of High Frequency Cochlear Hair Cells

doi: 10.1371/journal.pone.0013836

Figure Lengend Snippet: 3D renderings of confocal z-stacks of apical (A), middle (B), and basal (C) cochlear turns immunostained with a polyclonal antibody against the SK2 channel (green) show expression of the SK2 channel in the three rows of outer hair cells in all regions. Confocal z-stacks (23 optical sections) of basal cochlear turns immunostained with a polyclonal antibody against the SK2 channel (green, D) and monoclonal antibody against the BK channel (red, E) show colocalized expression (F) in the three rows of outer hair cells, further suggesting the localization of BK channels to the postsynaptic outer hair cell membrane. (Examination of single optical sections also shows colocalization of the SK2 and BK channel.) The size of the SK2 channel immunopuncta increases from apical to middle turns and then decreases in basal turns. Data are presented as box plots representing the median (box interior), the 25 th and 75 th percentile (box boundaries), the 10 th and 90 th percentile (whiskers), and the 5 th and 95 th percentile (dots). (G) 3D renderings of confocal z-stacks of basal cochlear turns immunostained with a polyclonal antibody against the SK2 channel (green), a monoclonal antibody against the BK channel (red), and a goat polyclonal antibody against synapsin (blue) confirm the colocalized expression of SK2 and BK channels in association with efferent terminals (H) in the three rows of outer hair cells. In basal turns, BK and SK2 channel immunoreactivity are colocalized. Grid dimensions equal 10 µm for top and bottom panels. Scale bar equals 10 µm for middle panels.

Article Snippet: Immunofluorescent staining was performed as described previously using the following antibodies: mouse monoclonal anti-BK (L6/23; 1∶500; provided by Dr. James Trimmer, UC Davis, CA), rabbit polyclonal anti BK (APC021; 1∶500; Alomone Labs, Jerusalem, Israel), rabbit polyclonal anti-SK2 (1∶500; provided by Dr John Adelman, Vollum Institute, University of Oregon Health Sciences), goat polyclonal anti-prestin (1∶300; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit polyclonal anti-synapsin (AB1543P; 1∶600; Chemicon/Millipore, Temecula, CA); and mouse monoclonal anti-Na-KATPase α3 subunit (MA3-915; 1∶600; Affinity Bioreagents, Rockford, IL).

Techniques: Expressing