Journal: Frontiers in Cellular Neuroscience

Article Title: The Endocannabinoid/Cannabinoid Receptor 2 System Protects Against Cisplatin-Induced Hearing Loss

doi: 10.3389/fncel.2018.00271

Figure Lengend Snippet: Distribution of the GFP-tagged CB2R knock-in mice. (A) The genetic background of the knock-in mice is shown where the GFP reporter gene was inserted within exon 3 of CB2R and the expression of GFP was driven by internal ribosomal entry (IRE) promoter. Cochleae isolated from these GFP-CB2R co-expressing knock-in mice were fixed in 4% paraformaldehyde followed by decalcification and then sectioned. (B) The representative mid-modiolar section shows endogenous GFP fluorescence (green) in different anatomical regions of the cochlea including organ of Corti (OC), stria vascularis (SV), spiral ligament (SL), and spiral ganglion (SG) neuron. (C) Immunolabeling of GFP-tagged CB2R (green) along with hair cell marker, Myo VIIa (red), show colocalization of CB2R in both, the OHCs and IHCs. (D) Co-labeling of these cochleae with Tuj1 (red), a neuronal marker, show the distribution of GFP-CB2R (green) in spiral ganglion neurons. (E) Whole mount sections of cochleae from GFP-CB2R co-expressing knock-in mice show co-localization of GFP-CB2R (green) and myosin VIIa (red) in the OHCs and IHCs of OC. (F) GFP-CB2R whole mounts sections were also co-stained with neuronal marker, Tuj1 (red), and pre-synaptic ribbon marker, CtBP2 (magenta), showing pre-synaptic localization of CB2R. Cell nuclei (blue) are stained with Hoechst stain in (B,E,F) . Studies reported here were repeated in at least three different animals and similar results were obtained.

Article Snippet: Two different myosin VIIa antibodies were used for different purposes: Mouse IgG1 anti-myosin-VIIa antibody was obtained from Developmental Studies Hybridoma Bank (#138-1), while rabbit anti-myosin-VIIa was purchased from Proteus Biosciences (#25-6790).

Techniques: Knock-In, Expressing, Isolation, Fluorescence, Immunolabeling, Marker, Labeling, Staining

Journal: Frontiers in Cellular Neuroscience

Article Title: The Endocannabinoid/Cannabinoid Receptor 2 System Protects Against Cisplatin-Induced Hearing Loss

doi: 10.3389/fncel.2018.00271

Figure Lengend Snippet: Distribution of the CB2R in the rat cochlea. (A) Immunolabeling of mid-modiolar cochlear sections with CB2R antibody shows labeling of cells in the organ of Corti (green). Labeling was observed in the outer hair cells (OHC), inner hair cells (IHC), inner pillar cell (IPC), and outer pillar cell (OPC). (B) Co-labeling of the organ of Corti with antibodies for CB2R (green) and myosin VIIa (red) showed co-localization of these proteins in the OHC and IHC. (C) Staining of CB2R was also observed in the intermediate and basal cells of the SV and type II fibrocytes in SL. CB2R (green) showed some overlap with Na + /K + -ATPase α1 subunit (red) in the SV. (D) CB2R was localized to the plasma membranes of SG. Approximately 60% of cells in this section were stained with both CB2R (green) and Hoechst (blue). (E) Co-labeling of CB2R and myosin VIIa in OHCs and IHCs in whole mount preparations of the organ of Corti shows that distribution of CB2R (green) is more widespread than that of myosin VIIa (red). Hoechst was used as a nuclear stain (blue) in (B–E) . Studies reported here were repeated in at least three different animals and similar results were obtained.

Article Snippet: Two different myosin VIIa antibodies were used for different purposes: Mouse IgG1 anti-myosin-VIIa antibody was obtained from Developmental Studies Hybridoma Bank (#138-1), while rabbit anti-myosin-VIIa was purchased from Proteus Biosciences (#25-6790).

Techniques: Immunolabeling, Labeling, Staining, Clinical Proteomics

Journal: Frontiers in Cellular Neuroscience

Article Title: The Endocannabinoid/Cannabinoid Receptor 2 System Protects Against Cisplatin-Induced Hearing Loss

doi: 10.3389/fncel.2018.00271

Figure Lengend Snippet: Trans -tympanic administration of JWH015 prevents cisplatin-induced hearing loss. (A) Baseline ABR thresholds were recorded in Wistar rats, which were then pre-treated with either trans -tympanic AM630 (2.5 nmoles) for 30 min followed by trans -tympanic JWH015 (2.5 nmoles) or individually with trans -tympanic AM630 or JWH015 for 24 h. Cisplatin (11 mg/kg) was then administered (i.p.) after day 1 and post-ABR was performed after 72 h. Pre-treatment with JWH015 diminished cisplatin-induce ABR threshold shift in all the three frequency regions. The protective action of JWH015 was completely blocked by AM630. Trans -tympanic administration of AM630 alone significantly elevated by ABR thresholds. Data (threshold shift) is plotted as mean ± SEM ( n = 12). (B) The animals were sacrificed at the end point and cochleae were collected, fixed by 4% PFA in PBS followed by decalcification for 3 weeks in 100 mM EDTA. The base of the cochlea were dissected out and stained with hair cell marker, myosin VIIa, to determine the hair cell loss. Representative images are shown. Scale bar is 20 μm. (C) The bar graph represents the average number of hair cells per 100 μm area shown in (B) ( n ≥ 3). Pre-treatment with JWH015 reduced cisplatin-induced outer hair cell loss. Administration of AM630 produced no hair cell loss. ∗ p < 0.05 vs. vehicle; ∗∗ p < 0.05 vs. cisplatin; # p < 0.05 vs. JWH015 + cisplatin; and ∗∗∗ p < 0.05 vs. JWH015 (one-way ANOVA).

Article Snippet: Two different myosin VIIa antibodies were used for different purposes: Mouse IgG1 anti-myosin-VIIa antibody was obtained from Developmental Studies Hybridoma Bank (#138-1), while rabbit anti-myosin-VIIa was purchased from Proteus Biosciences (#25-6790).

Techniques: Staining, Marker, Produced

Journal: Scientific Reports

Article Title: Fatty acid binding protein type 7 deficiency preserves auditory function in noise-exposed mice

doi: 10.1038/s41598-023-48702-4

Figure Lengend Snippet: Experimental design, body weights, and FABP7 expression in the cochlea. Schematic of the experiments using wild-type (WT) and Fabp7 knockout (KO) mice ( A ). The body weight (BW) of Fabp7 KO mice was not significantly different from that of WT mice ( B ). Immunohistochemistry of FABP7 in the cochlea of WT mice revealed that FABP7 was expressed throughout the spiral ganglion (SG), organ of Corti (OC), spiral limbus (SLim), and spiral ligament (SLig) ( C ). Scale bar, 200 μm ( C – D ). No expression of FABP7 was observed in the cochlea of Fabp7 KO mice ( D ). Maximum projection images of the OC showing high expression of FABP7 in Schwann cells and SOX2-positive supporting cells around the MYO7a-positive outer hair cells (OHCs) in the cochlea of WT mice ( E ). In the SG, FABP7 was expressed in satellite cells surrounding TUJ-1-positive neurons ( F ). Scale bars, 50 μm ( E – F ). Auditory brainstem response; ABR, weeks; W, post-noise exposure day; PNED, inner hair cell; IHC. Statistical significance was determined using two-way analysis of variance, followed by Šídák multiple comparison test. Error bars represent standard deviation.

Article Snippet: After rinsing with PBS, tissue sections were blocked with 3% bovine serum albumin/0.3% Triton X-100/ PBS for 30 min at room temperature, the unconjugated AffiniPure Fab fragment of anti-mouse IgG (1:10, Jackson ImmunoResearch, West Grove, PA) for 2 h at room temperature, and incubated overnight with the following primary antibodies: (1) rabbit IgG anti-FABP7 (1:1000, Merck, Darmstadt, Germany #PRS4259), (2) mouse IgG anti-TUJ-1 (1:500, Abcam, Cambridge, UK #ab78078), (3) mouse IgG2a anti-Myosin 7a (MYO7a) (1:200, Santa Cruz Biotechnology, Dallas, TX, USA #SC-74516), and (4) goat IgG anti-SOX2 (1:500, R&D Systems, Minneapolis, MN, USA #AF2018).

Techniques: Expressing, Knock-Out, Immunohistochemistry, Comparison, Standard Deviation

Journal: bioRxiv

Article Title: Relationship between vestibular hair cell loss and deficits in two anti-gravity reflexes in the rat

doi: 10.1101/2020.12.21.423804

Figure Lengend Snippet: Effect of ototoxic exposure on the density of sensory hair cells (HCs) in the vestibular epithelia. The example images correspond to the medial part of the utricle, peripheral to the striola. Type I HCs (HCI) are revealed by the Caspr+ label on the inner membrane of the calyx (green). Type II HCs (HCII) show no Caspr+ label but are Myo7a+ (red) and calretinin+ (white). Nuclei were labeled with DAPI (blue). Images shown here are stack projections optimized to provide an overall sense of HC density, not an accurate identification of the cell types. Cells that seem to be Myo7a+, Caspr- and calretinin-may be HCII that express calretinin at low levels or HCI with their calyceal junction in planes not included in the image, and Caspr1+ calyces with no visible Myo7a+ are HCI cells with suboptimal red mark in this particular image. Also, HC superposition may generate apparent calretinin label in Caspr1+ cells. For cell counts, color channels were split as shown in . (A) Normal density of cells in a control rat. (B) Control-like appearance was found in most rats receiving the lowest dose of IDPN (400 mg/kg). (C) Overt loss of HCI and HCII in an utricle of a rat dosed with 500 mg/kg of IDPN. (D) Complete loss of HCI and nearly complete loss of HCII in a rat dosed with 600 mg/kg of IDPN. Scale bars = 30 μm.

Article Snippet: The following primary antibodies were used: rabbit anti-Myosin VIIa (Myo7a) from Proteus Biosciences (cat. # 25-6790), rabbit anti-tenascin from Millipore (cat. # AB19013), rabbit anti-oncomodulin from Swant (cat. # OMG4), guinea pig anti-calretinin from Synaptic Systems (cat. # 214-104), mouse anti-Myo7a (clone 138-1-s, IgG1, supernatant) from Developmental Studies Hybridoma Bank, mouse anti-CtBP2/Ribeye (clone 16/CtBP2, IgG1, cat. # 612044) from BD Biosciences, mouse anti-contactin-associated protein (Caspr1) (clone K65/35, IgG1, cat. # 75-001) and anti-PSD95 (clone K28/43, IgG2a, cat# 75-028) from Neuromab.

Techniques: Labeling

Journal: bioRxiv

Article Title: Relationship between vestibular hair cell loss and deficits in two anti-gravity reflexes in the rat

doi: 10.1101/2020.12.21.423804

Figure Lengend Snippet: Differences in the loss of type I hair cells (HCI), type II hair cells (HCII), and all hair cells (HC) after exposure to the ototoxic compound, IDPN, as a function of the dose (400 to 1000 mg/kg, see legend), zone (central/striola vs periphery) and end-organ (utricle, crista, and saccule). The images in the left column show the use of the Caspr1 (green) and Myo7a (red) label to identify HCI, use of the Calretinin (white) and Myo7a (red) label to identify HCII and the use of Myo7a (red) label to identify all HCs. Nuclei in blue were labelled with DAPI. Scale bars = 10 μm. Bar graphs show superposed HCI and HCII counts (X±SE) according to end-organ, region within it and IDPN dose. The line show mean All HC counts. Error bars for All HCs are not included for clarity. The sum HCI+HCII and All HCs are not identical in all cases because the values correspond to counts obtained from separate images. Numbers below the bars indicate numbers of animals. #, *, +: p<0.05, significantly different from control group for HCII, HCI, and All HC, respectively, by Duncan’s test after significant ANOVA. Note that loss of HCI starts at low doses of IDPN, while these low doses have no effect on HCII counts, particularly in the saccule.

Article Snippet: The following primary antibodies were used: rabbit anti-Myosin VIIa (Myo7a) from Proteus Biosciences (cat. # 25-6790), rabbit anti-tenascin from Millipore (cat. # AB19013), rabbit anti-oncomodulin from Swant (cat. # OMG4), guinea pig anti-calretinin from Synaptic Systems (cat. # 214-104), mouse anti-Myo7a (clone 138-1-s, IgG1, supernatant) from Developmental Studies Hybridoma Bank, mouse anti-CtBP2/Ribeye (clone 16/CtBP2, IgG1, cat. # 612044) from BD Biosciences, mouse anti-contactin-associated protein (Caspr1) (clone K65/35, IgG1, cat. # 75-001) and anti-PSD95 (clone K28/43, IgG2a, cat# 75-028) from Neuromab.

Techniques: