Journal: Developmental biology

Article Title: Atoh1 directs hair cell differentiation and survival in the late embryonic mouse inner ear ☆

doi: 10.1016/j.ydbio.2013.06.022

Figure Lengend Snippet: Atoh1CreERT2/flox embryos have fewer myosin VIIa+ cells and fewer stereociliary bundles in the utricle. Pregnant dams were gavaged with tamoxifen at either E14.5 (A–F″) or E16.5 (G–L″) and utricles harvested at E18.5. Immunostaining for myosin VIIa (A, D, G, and J) and labeling with phalloidin (B, E, H, and K) revealed decreased mediolateral extent (arrows) of the hair cell field in Atoh1CreERT2/flox embryos (D–F, J–L) compared to control littermates (A–C, G–I). Yellow dotted lines in utricles from control littermates (A–C, G–I) show that hair cells with stereocilia are found right up to the border of the hair cell field. In contrast, red (D, F, J, and L) and green (E, F, K, and L) dotted lines in utricles from Atoh1CreERT2/flox embryos show that many peripherally-located hair cells lack stereocilia. Solid and dotted boxes indicate regions shown in A′–L′ and A″ –L″, respectively, and are representative of the locations of “central” and “peripheral” regions of interest for cell counts (see text). Fewer myosin VIIa+ hair cells are seen in these regions (A′, A″, D′, D″, G′, G″, J′, and J″), and more of these hair cells lack stereocilia in Atoh1CreERT2/flox embryos than in controls (B′–C″, E′–F″, H′–I″, and K′–L″). Examples of long (l), short (s) and absent (a) stereociliary bundles are shown in K′–L″. Immunostaining for β-galactosidase (M) shows that Atoh1CreERT2/flox; ROSALacZ embryos have many cells that have undergone recombination, but only a small number of these have phalloidin+ hair bundles (N, O). Scale bar: 30 μm (A–L); 12 μm (M–O); 10 μm (A′–L″).

Article Snippet: Whole cochleae or whole utricles were blocked for 30 min at room temperature (RT) in 1X PBS/ 0.3% Triton X-100/3% non-fat dry milk (PBST-NFDM) (cochleae) or 2% bovine serum albumin/0.8% normal goat serum/0.5% Triton X-100 (utricles) and incubated at 4 °C overnight in the same solution containing primary antibodies at the following dilutions: rabbit anti-myosin VI (25–6790, Proteus Bioscience) 1:500; rabbit anti-myosin VIIa (Proteus Biosciences, Ramona, CA) 1:500; chicken anti-β-galactosidase (ab9361, Abcam) 1:500; Alexa Fluor 488-conjugated phalloidin (A12379, Invitrogen) 1:100; Alexa Fluor 647-conjugated phalloidin ( {"type":"entrez-nucleotide","attrs":{"text":"A22287","term_id":"641467","term_text":"A22287"}} A22287 , Invitrogen) 10 μg/ml; rabbit anti-cleaved caspase 3 (9661S, Cell Signaling) 1:100.

Techniques: Immunostaining, Labeling, Control

Journal: Developmental biology

Article Title: Atoh1 directs hair cell differentiation and survival in the late embryonic mouse inner ear ☆

doi: 10.1016/j.ydbio.2013.06.022

Figure Lengend Snippet: Utricular hair cells require Atoh1 for survival and stereocilia formation. Total numbers of immunostained cells were counted in the indicated number of utricles harvested from E18.5 embryos and reported as average number ± SEM. Reported decreases are relative to littermate controls. Values for E14.5 and E16.5 tamoxifen administration are compared within genotype at the bottom of the Table.

Article Snippet: Whole cochleae or whole utricles were blocked for 30 min at room temperature (RT) in 1X PBS/ 0.3% Triton X-100/3% non-fat dry milk (PBST-NFDM) (cochleae) or 2% bovine serum albumin/0.8% normal goat serum/0.5% Triton X-100 (utricles) and incubated at 4 °C overnight in the same solution containing primary antibodies at the following dilutions: rabbit anti-myosin VI (25–6790, Proteus Bioscience) 1:500; rabbit anti-myosin VIIa (Proteus Biosciences, Ramona, CA) 1:500; chicken anti-β-galactosidase (ab9361, Abcam) 1:500; Alexa Fluor 488-conjugated phalloidin (A12379, Invitrogen) 1:100; Alexa Fluor 647-conjugated phalloidin ( {"type":"entrez-nucleotide","attrs":{"text":"A22287","term_id":"641467","term_text":"A22287"}} A22287 , Invitrogen) 10 μg/ml; rabbit anti-cleaved caspase 3 (9661S, Cell Signaling) 1:100.

Techniques:

Journal: Developmental biology

Article Title: Atoh1 directs hair cell differentiation and survival in the late embryonic mouse inner ear ☆

doi: 10.1016/j.ydbio.2013.06.022

Figure Lengend Snippet: Atoh1 CreERT2/fox embryonic utricles have regionally-variable differentiation defects but no change in cell density. Pregnant dams received tamoxifen at E14.5 and embryos were harvested at E18.5. Total numbers of DAPI+ nuclei in the regions of interest (450−500 cells; see Methods) were counted for cell density calculations (DAPI+nuclei/100 μm 2 ). Hair bundles were qualitatively scored as long, short or absent. All numbers are reported as average ± SEM; percent change is relative to littermate controls.

Article Snippet: Whole cochleae or whole utricles were blocked for 30 min at room temperature (RT) in 1X PBS/ 0.3% Triton X-100/3% non-fat dry milk (PBST-NFDM) (cochleae) or 2% bovine serum albumin/0.8% normal goat serum/0.5% Triton X-100 (utricles) and incubated at 4 °C overnight in the same solution containing primary antibodies at the following dilutions: rabbit anti-myosin VI (25–6790, Proteus Bioscience) 1:500; rabbit anti-myosin VIIa (Proteus Biosciences, Ramona, CA) 1:500; chicken anti-β-galactosidase (ab9361, Abcam) 1:500; Alexa Fluor 488-conjugated phalloidin (A12379, Invitrogen) 1:100; Alexa Fluor 647-conjugated phalloidin ( {"type":"entrez-nucleotide","attrs":{"text":"A22287","term_id":"641467","term_text":"A22287"}} A22287 , Invitrogen) 10 μg/ml; rabbit anti-cleaved caspase 3 (9661S, Cell Signaling) 1:100.

Techniques:

Journal: Redox Biology

Article Title: Targeting nitrative stress for attenuating cisplatin-induced downregulation of cochlear LIM domain only 4 and ototoxicity

doi: 10.1016/j.redox.2016.10.016

Figure Lengend Snippet: Cisplatin-induced increase in nitrotyrosine is attenuated by SRI110. A) Increased levels of nitrotyrosine as well as condensed nuclei (white arrows) were detected in UBOC1 cells treated with 10 µm cisplatin. Co-treatment with 50 µm SRI110 attenuated the cisplatin-induced increase in nitrotyrosine. Red staining indicates immunoreactivity to anti-nitrotyrosine, blue indicates nuclear staining with DAPI, while green indicates actin staining with phalloidin. The images are representative of four biological replicates. Scale bar, 20 µm. (B) The mean pixel intensities for nitrotyrosine immunostaining was significantly higher in cisplatin treated cells (****P<0.0001), which was attenuated by SRI110 co-treatment (***P<0.001). The results are expressed as mean±standard deviation, n=4. (C) Immunostaining (violet) with anti-myosin VIIa indicated the differentiated state of UBOC1 cells. The image is representative of three biological replicates. Scale bar, 20 µm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Then the cells were incubated with anti-nitrotyrosine, anti-myosin VIIa (catalog no. sc-32757, sc-74516, Santa Cruz Biotechnology Inc., Santa Cruz, CA) or anti-LMO4 (catalog no. ab39383, Abcam, Cambridge, MA) followed by incubation with Alexa Fluor 568 donkey anti-mouse or Alexa Fluor 647 goat anti-rabbit secondary antibody (catalog no. A10037 or A21244, Life Technologies, Carlsbad, CA) and fluorescein phalloidin (catalog no. F432, Life Technologies).

Techniques: Staining, Immunostaining, Standard Deviation

Journal: Journal of Cell Science

Article Title: Caprin-1 is a target of the deafness gene Pou4f3 and is recruited to stress granules in cochlear hair cells in response to ototoxic damage

doi: 10.1242/jcs.076141

Figure Lengend Snippet: Caprin-1 is expressed in the inner ear in both supporting cells and hair cells. (A) Levels of Caprin-1 and Pou4f3 mRNA were determined by qPCR in mouse cochlea, utricle and brain. For all qPCR experiments, 18S rRNA was used as an endogenous control and expression levels were calculated relative to this endogenous control. Error bars represent 95% confidence intervals. (B) Schematic showing the arrangement of cells within the organ of Corti. OH, outer hair cells; IH, inner hair cells; D, Deiters' cells; H, Hensen's cells; C, Claudius' cells; P, pillar cells; and Ph, phalangeal cells. (C) Vibratome slices through a P2 rat cochlea show Caprin-1 (green) expression in cells in the sensory epithelium (the organ of Corti), the hair cell marker myosin VIIa (red) and nuclei stained with DAPI (blue). Arrowheads indicate greater Caprin-1 expression in phalangeal processes of the Deiters' cells that intercalate between hair cells. The asterisks indicate expression in Claudius' cells. Scale bar: 10 μm. (D) Samples enriched for particular cell types (supporting-cell-enriched, hair-cell-enriched and a mixed-cell sample of Deiters' and hair cells) were collected and analysed by qPCR. Expression levels of p27kip1, Myo7a, Pou4f3 and Caprin-1 were calculated relative to the endogenous control. p27kip1 and Myo7a levels indicate the degree of enrichment for supporting cells and hair cells, respectively. Pou4f3 expression is highest in the hair-cell-enriched pool, whereas Caprin-1 was lowest in the hair-cell-enriched pool. (E) Single-cell qPCR. Caprin-1 levels were measured in individual hair cell samples (X, Y and Z) that expressed Myo7a but not p27kip1. These cells were collected from the same explant culture, but are representative of results obtained from more than five cultures.

Article Snippet: Antibodies were against the following proteins: Caprin-1 (rabbit, 1:200) ( Solomon et al., 2007 ); TIA-1 (C-20) (goat polyclonal antibody, 1:200; Santa Cruz Biotechnology); myosin VIIa (138-1) [mouse, 1:250; developed by Soni et al. ( Soni et al., 2005 ) and obtained from the Developmental Studies Hybridoma Bank (Department of Biology, University of Iowa, Iowa City, IA)].

Techniques: Control, Expressing, Marker, Staining

Journal: Journal of Cell Science

Article Title: Caprin-1 is a target of the deafness gene Pou4f3 and is recruited to stress granules in cochlear hair cells in response to ototoxic damage

doi: 10.1242/jcs.076141

Figure Lengend Snippet: Changes in TIA-1 and Caprin-1 expression during neomycin treatment. Cochlear explant cultures from P2 rats were treated with 1 mM neomycin for 0, 3, 6 or 12 hours. Caprin-1, TIA-1 and myosin VIIa expression were determined by triple-label immunofluorescence, with DAPI to assess chromatin structure. Areas shown are from the basal end of cochlear coils and were selected without observing fluorescent signals, to avoid bias. Images are the averages of four confocal optical sections representing ~6-μm-optical-slices of the outer hair cell region. The upper and lower rows represent focal planes of the upper apical pole and lower nuclear pole of hair cells, respectively (see inset). Arrowheads indicate hair cell granules labelled with both Caprin-1 and TIA-1 (also see supplementary material Fig. S2). Asterisks indicate condensed nuclei. Scale bar: 10 μm (for all images).

Article Snippet: Antibodies were against the following proteins: Caprin-1 (rabbit, 1:200) ( Solomon et al., 2007 ); TIA-1 (C-20) (goat polyclonal antibody, 1:200; Santa Cruz Biotechnology); myosin VIIa (138-1) [mouse, 1:250; developed by Soni et al. ( Soni et al., 2005 ) and obtained from the Developmental Studies Hybridoma Bank (Department of Biology, University of Iowa, Iowa City, IA)].

Techniques: Expressing, Immunofluorescence

Journal: Frontiers in Cellular Neuroscience

Article Title: Age-related alterations in efferent medial olivocochlear-outer hair cell and primary auditory ribbon synapses in CBA/J mice

doi: 10.3389/fncel.2024.1412450

Figure Lengend Snippet: Antibody labeling.

Article Snippet: Anti-MyosinVIIa (1:200) , HIS-tagged synthetic peptide in the human MYO7A sequence (a.a. 927–1,203) , DSHB** – mouse monoclonal , MYO7A 138–1 RRID: AB_2282417 , Immunohistochemistry, immunoblot/Western blot ( ) , Hair cells ( ; ; ) .

Techniques: Labeling, Staining, Recombinant, Expressing, Marker, Sequencing, Immunohistochemistry, Western Blot, Immunoprecipitation, Membrane

Journal: The Journal of Cell Biology

Article Title: Wnts regulate planar cell polarity via heterotrimeric G protein and PI3K signaling

doi: 10.1083/jcb.201912071

Figure Lengend Snippet: Growth defects in Wls cKO cochleae. (a) Schematic diagrams of a cochlear duct (CD) cross-section (top), and en face view of the OC and surrounding nonsensory epithelia (bottom). HCs are colored green and SCs magenta. (a’) A schematic diagram of the HC apical cytoskeleton. The medially polarized microvilli zone is shown in gray. (b–e) Cross-sections of E16.5 control (b and d) and Wls cKO cochleae (c and e) stained for Wls (magenta), Tuj1 (green), and nuclei (blue). (f and g) Paint-filled control (f) and Wls cKO (g) inner ears at E14.5. (h and i) Dissected E18.5 control (h) and Wls cKO cochleae (i). (j and k) Quantifications of cochlear length and HC numbers. n = 6 for each genotype. ***, P < 0.001. (l–o) Confocal images of E18.5 control (l and n) and Wls cKO OC (m and o) stained for myosin VIIa and Sox2. Arrowheads indicate the pillar cell row. Scale bars: b–e, 100 µm; f and g, 250 µm; h and i, 100 µm; l–o, 6 µm. Co, cochlea; LER, lesser epithelial ridge; SG, spiral ganglion.

Article Snippet: Anti-myosin VIIa , 1:500 , Proteus BioSciences , , 25-6790.

Techniques: Control, Staining

Journal: Nature Communications

Article Title: EMX2-GPR156-Gαi reverses hair cell orientation in mechanosensory epithelia

doi: 10.1038/s41467-021-22997-1

Figure Lengend Snippet: a LacZ reporter is expressed throughout the sensory region in Gpr156 del/+ vestibular organs (ant/lat c., anterior/lateral crista). b LacZ expression is limited to MYO7A + HCs in a saccule cross-section. X-gal signal is trapped in HC vesicles (arrow in magnified inset) but support cells (arrowhead) are negative. c , d P2 wild-type utricle where basal body labeling (PCNT) indicates HC orientation. GPR156 polarization (solid arrowheads) is limited to lateral HCs oriented medially. HCs across the LPR oriented laterally do not show polarized GPR156 (hollow arrowheads). Boxed regions in continuous fields in the left panels are magnified in the central and right panels (saccule: see Supplementary Fig. ). d GPR156 enrichment in the utricle LPR domain at the HC junction opposite (opp. BB) or near (BB) the basal body. HCs oriented medially (left) are analyzed separately from HC oriented laterally (right). GPR156 is expressed as ratio of ZO1 signal (mean ± SD; n , HC numbers in 3 animals; Kruskal-Wallis test with Dunn’s multiple comparisons, **** p < 0.0001; * p = 0.0332). e Summary of HC orientation (arrows), GPR156 protein distribution (magenta) and previously reported Emx2 expression (blue) by vestibular organ in normal and mutant conditions. The scheme in c indicates the position of the domain analyzed in c and d (blue). Scale bars are 100 µm ( a ), 50 µm ( b ), 20 µm ( c ).

Article Snippet: Primary antibodies used were: goat anti-GPR156 (Santa Cruz; sc102572; TCA; 1:100), rabbit anti-GPR156 (Novus; NBP1-83402; TCA; 1:100), mouse anti-acetylated Tubulin (Santa Cruz; 23950; PFA; 1:500), rabbit anti-pericentrin/PCNT (Biolegend; PRB-432C; PFA; 1:400), mouse anti-βII-Spectrin/SPTBN2 (BD Transduction Lab; 612562, PFA; 1:200), rat anti-ZO1 (Developmental Studies Hybridoma Bank; R26.4C; TCA; 1:200), rabbit anti-Gαi3 (Santa Cruz; sc-262; PFA; 1:400), chicken anti-Gαi3 (Sigma; GW22489; PFA; 1:400; used for cochlear explants), rabbit anti-DAPLE/CCDC88C (Proteintech; 25769-1-AP; TCA; 1:400), mouse anti-MYO7A (Developmental Studies Hybridoma Bank; 138-1; PFA; 1:500), goat anti-FZD6 (R&D Systems; AF1526; PFA; 1:200), rabbit anti-VANGL2 (gift from Philippe Gros, McGill University; PFA; 1:500), goat anti-GPSM2/LGN (ThermoFisher Scientific; PA5-18646; PFA; 1:200), rabbit anti-βGalactosidase (Cappel discontinued aliquot; PFA; 1:1000; now MP Biomedical 55976), mouse anti-aPKC/PRKCZ (Santa Cruz; sc-216; PFA; 1:100), rabbit anti-PARD6B (Santa Cruz; sc-67393; PFA; 1:100), rabbit anti-PARD3A (Proteintech; 11085-1-AP; PFA; 1:200), rabbit anti-CALB1 (Cedarlane/Millipore; AB1778(CH); PFA; 1:500), goat anti-SPP1/Osteopontin (R&D Systems; AF808; PFA; 1:100), goat anti-SOX2 (Santa Cruz; 17320; PFA; 1:500), rabbit anti-EMX2 (Trans Genic; KO609; PFA; 1:250).

Techniques: Expressing, Labeling, Mutagenesis

Journal: Nature Communications

Article Title: EMX2-GPR156-Gαi reverses hair cell orientation in mechanosensory epithelia

doi: 10.1038/s41467-021-22997-1

Figure Lengend Snippet: a LPR domain in E17.5 utricles. Polarization of GPR156 in lateral HCs (arrowhead) is lost when EMX2 is missing and these HCs fail to reverse. b GPR156 enrichment in the lateral utricle (LES) domain. c Medial domain in E18.5 utricles. Ectopic expression of Emx2 reverses HC orientation and induces polarization of GPR156 (arrowhead) in medial HCs. d GPR156 enrichment in the utricle medial domain. e LPR domain in E18.5 utricles. Polarization of GPR156 in lateral HCs expressing PTXa is intact although these HCs fail to reverse. Utricles are labeled with GPR156, PCNT, and ZO1 ( a , c ) or MYO7A ( e ). In a , c , e boxed areas are magnified in the lower panels, and HC orientation and GPR156 distribution are summarized in a cartoon form. In b , d GPR156 enrichment is measured at the junction opposite (opp. BB) or near (BB) the basal body in the same HC. GPR156 is expressed as ratio of ZO1 signal (mean ± SD; n, HC numbers in 3 or more animals; Kruskal-Wallis test, **** p < 0.0001, ns p > 0.9999). Arrows indicate HC orientation based on PCNT-labeled basal body. Utricle schemes indicate the domain imaged or analyzed (blue). Yellow dashed lines represent the LPR in controls. See Supplementary Fig. for related saccule and crista results. Scale bars are 10 µm ( a , c ), 20 µm ( e ).

Article Snippet: Primary antibodies used were: goat anti-GPR156 (Santa Cruz; sc102572; TCA; 1:100), rabbit anti-GPR156 (Novus; NBP1-83402; TCA; 1:100), mouse anti-acetylated Tubulin (Santa Cruz; 23950; PFA; 1:500), rabbit anti-pericentrin/PCNT (Biolegend; PRB-432C; PFA; 1:400), mouse anti-βII-Spectrin/SPTBN2 (BD Transduction Lab; 612562, PFA; 1:200), rat anti-ZO1 (Developmental Studies Hybridoma Bank; R26.4C; TCA; 1:200), rabbit anti-Gαi3 (Santa Cruz; sc-262; PFA; 1:400), chicken anti-Gαi3 (Sigma; GW22489; PFA; 1:400; used for cochlear explants), rabbit anti-DAPLE/CCDC88C (Proteintech; 25769-1-AP; TCA; 1:400), mouse anti-MYO7A (Developmental Studies Hybridoma Bank; 138-1; PFA; 1:500), goat anti-FZD6 (R&D Systems; AF1526; PFA; 1:200), rabbit anti-VANGL2 (gift from Philippe Gros, McGill University; PFA; 1:500), goat anti-GPSM2/LGN (ThermoFisher Scientific; PA5-18646; PFA; 1:200), rabbit anti-βGalactosidase (Cappel discontinued aliquot; PFA; 1:1000; now MP Biomedical 55976), mouse anti-aPKC/PRKCZ (Santa Cruz; sc-216; PFA; 1:100), rabbit anti-PARD6B (Santa Cruz; sc-67393; PFA; 1:100), rabbit anti-PARD3A (Proteintech; 11085-1-AP; PFA; 1:200), rabbit anti-CALB1 (Cedarlane/Millipore; AB1778(CH); PFA; 1:500), goat anti-SPP1/Osteopontin (R&D Systems; AF808; PFA; 1:100), goat anti-SOX2 (Santa Cruz; 17320; PFA; 1:500), rabbit anti-EMX2 (Trans Genic; KO609; PFA; 1:250).

Techniques: Expressing, Labeling

Journal: Nature Communications

Article Title: EMX2-GPR156-Gαi reverses hair cell orientation in mechanosensory epithelia

doi: 10.1038/s41467-021-22997-1

Figure Lengend Snippet: a Schematic of the lateral-line system in a 5 day-post-fertilization zebrafish. Neuromast HCs show binary orientation along the antero-posterior (A–P) or dorso-ventral (D–V) axis as depicted. Emx2 is only expressed in HCs of one orientation in each neuromast (green D to V and A to P HCs). HC orientation is indicated by a black dot representing the off-center basal body. b – i Phalloidin labeling in neuromasts reveals HC orientation by the lack of signal above the off-center basal body. In wild-type siblings ( b , e , f , i ), neuromasts contain an equal proportion of HCs with either orientation. In gpr156 mutants ( c – e , g – i ), there are more P to A ( c – e ) and V to D ( g – i )-oriented HCs compared to wild-type siblings (Tukey’s multiple comparison test, P to A exon2 allele p < 0.0001, sa34566 allele p < 0.0001; V to D exon2 allele p < 0.0001, sa34566 allele p < 0.0001). Green and blue asterisks highlight the two HC orientations in wild-type sibling neuromasts. Magenta and yellow asterisks highlight outlier HCs oriented 180° or 90° compared to the majority of HCs in gpr156 mutants. n = 10 neuromasts and N ≥ 8 animals per genotype, examined at 5 dpf. j , k Emx2 and Myo7a co-labeling in neuromasts. Wild-type siblings and gpr156 mutants neuromasts have a similar number of HCs ( l ) (mean ± SEM; unpaired t-test (two-tailed), A-P p = 0.1686; Mann–Whitney test (two-tailed), D-V p = 0.8547), and a similar proportion of HCs express Emx2 per neuromast ( m ) (mean ± SEM; unpaired t-test (two-tailed), A-P p = 0.5756; Mann–Whitney test (two-tailed), D-V p = 0.4805). In l – m the number of neuromasts (n) examined at 5 dpf in N ≥ 8 animals per genotype is indicated. n Scheme showing the GCaMP6s calcium reporter (blue and green) and the imaging plane in a neuromast. o1 , p1 Baseline gray scale GCaMP6s images of the hair bundle imaging plane in wild-type siblings ( o1 ) and gpr156 mutants ( p1 ; sa34566 allele). o2 , o3 , p2 , p3 Spatial patterns of GCaMP6s calcium signal increases in hair bundles during P to A ( o2 , p2 ) or A to P ( o3 , p3 ) directed fluid-jet stimulation. GCaMP6s signals are colorized according to the ∆F heat maps and superimposed onto prestimulus (prestim) baseline images ( o1 , p1 ). q In wild-type siblings, GCaMP6s signals are detected during both P to A and A to P directed stimulation ( o2 , o3 ). In contrast, compared to wild-type, in gpr156 mutants, significantly more hair bundles respond to P to A directed stimulation ( p2 – p3 ) (Sidak’s multiple comparison test, P to A p = 0.0008; n = 8 neuromasts per genotype and N = 4 wild-type and N = 3 mutant animals, examined at 5 dpf. See Supplementary Fig. for individual HC responses). NM, neuromast; sib, wild-type sibling. Scale bars are 5 µm ( b – d and f – h , j , k , o 1 – 3 , and p 1 – p 3 ).

Article Snippet: Primary antibodies used were: goat anti-GPR156 (Santa Cruz; sc102572; TCA; 1:100), rabbit anti-GPR156 (Novus; NBP1-83402; TCA; 1:100), mouse anti-acetylated Tubulin (Santa Cruz; 23950; PFA; 1:500), rabbit anti-pericentrin/PCNT (Biolegend; PRB-432C; PFA; 1:400), mouse anti-βII-Spectrin/SPTBN2 (BD Transduction Lab; 612562, PFA; 1:200), rat anti-ZO1 (Developmental Studies Hybridoma Bank; R26.4C; TCA; 1:200), rabbit anti-Gαi3 (Santa Cruz; sc-262; PFA; 1:400), chicken anti-Gαi3 (Sigma; GW22489; PFA; 1:400; used for cochlear explants), rabbit anti-DAPLE/CCDC88C (Proteintech; 25769-1-AP; TCA; 1:400), mouse anti-MYO7A (Developmental Studies Hybridoma Bank; 138-1; PFA; 1:500), goat anti-FZD6 (R&D Systems; AF1526; PFA; 1:200), rabbit anti-VANGL2 (gift from Philippe Gros, McGill University; PFA; 1:500), goat anti-GPSM2/LGN (ThermoFisher Scientific; PA5-18646; PFA; 1:200), rabbit anti-βGalactosidase (Cappel discontinued aliquot; PFA; 1:1000; now MP Biomedical 55976), mouse anti-aPKC/PRKCZ (Santa Cruz; sc-216; PFA; 1:100), rabbit anti-PARD6B (Santa Cruz; sc-67393; PFA; 1:100), rabbit anti-PARD3A (Proteintech; 11085-1-AP; PFA; 1:200), rabbit anti-CALB1 (Cedarlane/Millipore; AB1778(CH); PFA; 1:500), goat anti-SPP1/Osteopontin (R&D Systems; AF808; PFA; 1:100), goat anti-SOX2 (Santa Cruz; 17320; PFA; 1:500), rabbit anti-EMX2 (Trans Genic; KO609; PFA; 1:250).

Techniques: Labeling, Two Tailed Test, MANN-WHITNEY, Imaging, Mutagenesis

Journal: Aging (Albany NY)

Article Title: Down-regulation of Cav1.3 in auditory pathway promotes age-related hearing loss by enhancing calcium-mediated oxidative stress in male mice

doi: 10.18632/aging.102203

Figure Lengend Snippet: Age-related Cav1.3 expression in cochlea. ( A , B ) immunofluorescence of CaV1.3(green) and Myo7a (red) in the organ of Corti (left) and spiral ganglion (right) (magnification, ×400), nuclei was visualized by DAPI (blue). ( C ) the immunofluorescent staining for CaV1.3 (green) in the whole cochlear basilar membrane. ( D ) quantitative analysis of CaV1.3 expression in hair cells, spiral ganglion and cochlea basilar membrane.

Article Snippet: After overnight incubation with the primary antibody, rabbit anti-CaV1.3 calcium channel polyclonal antibody (1:50; Alomone labs, Israel) or mouse anti-Myo7a antibody (1:100; Santa Cruz, CA) at 4°C the sections were washed three times with PBS and incubated using Dylight 488 conjugated goat anti-rabbit IgG or Dylight 594 conjugated goat anti-mouse (1:500; Multi-Sciences, Hangzhou, China) for 1 h at room temperature.

Techniques: Expressing, Immunofluorescence, Staining, Membrane