rabbit anti gjb2  (Alomone Labs)

Journal: Molecules

Article Title: Connexin Expression in Human Minor Salivary Glands: An Immunohistochemical Microscopy Study

doi: 10.3390/molecules27185926

Figure Lengend Snippet: IF analysis for Cx26, Cx32, and Cx43 in NS-hLSGBs. ( A – C ) Representative confocal microscopy images of double IF with transmitted light contrast. Merged images show Cx positivity in red, ASMA positivity in green, and Cx-ASMA colocalization in yellow. These images have been modified by enhancing the saturation in order to better appreciate the yellow stain. White arrows indicate red immunoreaction at the plasma membrane of the duct cells and at the basal/basolateral side of the acinar cells. Black arrows point to the yellow spot due to the colocalization of Cx and ASMA. Inserts show the higher magnification of the squares. ( D – F ) Single IF images representing Cx immunoreactions (red) corresponding to ( A – C ) microscopic fields. A, acinus; D, duct. Scale bars, 20 µm; Inserts, 10 µm.

Article Snippet: Primary antibodies were as follows: rabbit anti-human Cx26 antibody (1:100, RαCx26, NBP2-41304, Novus Biologicals, Abingdon, UK), rabbit anti-human Cx32 antibody (1:25, RαCx32, Invitrogen 71-0600, Waltham, MA, USA), rabbit anti-human Cx43 antibody (1:100, RαCx43, NB1000-91717, Novus Biologicals, Abingdon, UK), and mouse anti-human ASMA (1:400, A2547, Sigma Aldrich, Saint Louis, MO, USA).

Techniques: Confocal Microscopy, Modification, Staining, Clinical Proteomics, Membrane

Journal: Molecules

Article Title: Connexin Expression in Human Minor Salivary Glands: An Immunohistochemical Microscopy Study

doi: 10.3390/molecules27185926

Figure Lengend Snippet: IF analysis for Cx26, Cx32, and Cx43 in SS-hLSGBs. ( A – C ) Representative confocal microscopy images of double IF with transmitted light contrast. Merged images show Cx positivity in red, ASMA positivity in green, and Cx-ASMA colocalization in yellow. These images have been modified by enhancing the saturation in order to better appreciate the yellow stain. White arrows indicate red immunoreaction at the plasma membrane of the duct cells and at the basal/basolateral side of the acinar cells. Black arrows point to the yellow spot due to the colocalization of Cx and ASMA. Inserts show the higher magnification of the squares. ( D – F ) Single IF images representing Cx immunoreactions (red) corresponding to ( A – C ) microscopic fields. Asterisks (*) point to the inflammatory cells. A, acinus; D, duct. Scale bars, 20 µm.

Article Snippet: Primary antibodies were as follows: rabbit anti-human Cx26 antibody (1:100, RαCx26, NBP2-41304, Novus Biologicals, Abingdon, UK), rabbit anti-human Cx32 antibody (1:25, RαCx32, Invitrogen 71-0600, Waltham, MA, USA), rabbit anti-human Cx43 antibody (1:100, RαCx43, NB1000-91717, Novus Biologicals, Abingdon, UK), and mouse anti-human ASMA (1:400, A2547, Sigma Aldrich, Saint Louis, MO, USA).

Techniques: Confocal Microscopy, Modification, Staining, Clinical Proteomics, Membrane

Journal: Molecules

Article Title: Connexin Expression in Human Minor Salivary Glands: An Immunohistochemical Microscopy Study

doi: 10.3390/molecules27185926

Figure Lengend Snippet: TEM immunogold analysis for Cx26 in NS-hLSGB. Representative images of immunoreactions (arrows) at the level of different cell compartments: rough endoplasmic reticulum ( A ), Golgi complex ( B ), and autophagic vacuole ( C ) in mucous cells; mitochondrial inner membrane and cristae in mucous ( D ) and serous ( E ) cells; nucleus ( F ) and apparent GJ in the lateral plasma membranes between the mucous cells ( G ). In ( E ), gold particles are also visible on the plasma membrane of a myoepitelial cell (MEC) bordering the serous cell. In ( H ), immunogold reaction is on an apparent GJ between the lateral plasma membranes of two duct cells. D, desmosome; GC, Golgi complex; L, lumen; M, mitochondrium; MEC, myoepithelial cell; MSG, mucous secretory granule; N, nucleus; RER, rough endoplasmic reticulum; SSG, serous secretory granule. Scale bars ( A – D ) and ( F – H ), 200 nm; ( E ) 500 nm.

Article Snippet: Primary antibodies were as follows: rabbit anti-human Cx26 antibody (1:100, RαCx26, NBP2-41304, Novus Biologicals, Abingdon, UK), rabbit anti-human Cx32 antibody (1:25, RαCx32, Invitrogen 71-0600, Waltham, MA, USA), rabbit anti-human Cx43 antibody (1:100, RαCx43, NB1000-91717, Novus Biologicals, Abingdon, UK), and mouse anti-human ASMA (1:400, A2547, Sigma Aldrich, Saint Louis, MO, USA).

Techniques: Membrane, Clinical Proteomics

Journal: Molecules

Article Title: Connexin Expression in Human Minor Salivary Glands: An Immunohistochemical Microscopy Study

doi: 10.3390/molecules27185926

Figure Lengend Snippet: TEM immunogold analysis for Cx26 ( A , F ), Cx32 ( B , D , G ), and Cx43 ( C , E , H ) in NS-hLSGB myoepithelial cells. Immunoreactions (arrows) are visible at the level of the nucleus ( A , D , E ), the basal plasma membrane ( A – C ), among fibrils ( F – H ), and on the mitochondria ( H ). BL, basal lamina; M, mitochondrion; N, nucleus. Scale bars ( A , B , F , H ), 200 nm; ( C – E , G ) 500 nm.

Article Snippet: Primary antibodies were as follows: rabbit anti-human Cx26 antibody (1:100, RαCx26, NBP2-41304, Novus Biologicals, Abingdon, UK), rabbit anti-human Cx32 antibody (1:25, RαCx32, Invitrogen 71-0600, Waltham, MA, USA), rabbit anti-human Cx43 antibody (1:100, RαCx43, NB1000-91717, Novus Biologicals, Abingdon, UK), and mouse anti-human ASMA (1:400, A2547, Sigma Aldrich, Saint Louis, MO, USA).

Techniques: Clinical Proteomics, Membrane

Journal: Molecules

Article Title: Connexin Expression in Human Minor Salivary Glands: An Immunohistochemical Microscopy Study

doi: 10.3390/molecules27185926

Figure Lengend Snippet: RT-PCR analysis for Cx26, Cx32, and Cx43 in NS-hLSGBs. Example of the threshold cycle (Ct), relative amplification curves, and Melting peak (the negative first derivative of the change in the fluorescence plotted as a function of temperature) for: Cx43 (blue) ( A , B ), Cx32 (red) ( C , D ), and Cx26 (green) ( E , F ); relative expression of Cxs in NS-hLSGB; data are expressed as means ± SEM ( G ).

Article Snippet: Primary antibodies were as follows: rabbit anti-human Cx26 antibody (1:100, RαCx26, NBP2-41304, Novus Biologicals, Abingdon, UK), rabbit anti-human Cx32 antibody (1:25, RαCx32, Invitrogen 71-0600, Waltham, MA, USA), rabbit anti-human Cx43 antibody (1:100, RαCx43, NB1000-91717, Novus Biologicals, Abingdon, UK), and mouse anti-human ASMA (1:400, A2547, Sigma Aldrich, Saint Louis, MO, USA).

Techniques: Reverse Transcription Polymerase Chain Reaction, Amplification, Fluorescence, Expressing

Journal: Molecules

Article Title: Connexin Expression in Human Minor Salivary Glands: An Immunohistochemical Microscopy Study

doi: 10.3390/molecules27185926

Figure Lengend Snippet: Details of gene specific primers used in real-time PCR experiments.

Article Snippet: Primary antibodies were as follows: rabbit anti-human Cx26 antibody (1:100, RαCx26, NBP2-41304, Novus Biologicals, Abingdon, UK), rabbit anti-human Cx32 antibody (1:25, RαCx32, Invitrogen 71-0600, Waltham, MA, USA), rabbit anti-human Cx43 antibody (1:100, RαCx43, NB1000-91717, Novus Biologicals, Abingdon, UK), and mouse anti-human ASMA (1:400, A2547, Sigma Aldrich, Saint Louis, MO, USA).

Techniques: Real-time Polymerase Chain Reaction, Sequencing, Amplification

Journal: bioRxiv

Article Title: Cell-specific delivery of GJB2 restores auditory function in mouse models of DFNB1 deafness and mediates appropriate expression in NHP cochlea

doi: 10.1101/2024.12.24.630240

Figure Lengend Snippet: a. Schematics of the AAV vectors used in the study. All vectors are single stranded. ITR: inverted terminal repeat, CBA: hybrid CMV enhancer/chicken β-actin promoter, HA: hemagglutinin tag, WPRE: woodchuck hepatitis virus post-transcriptional regulatory element, BGH: bovine growth hormone poly A signal. b. Survival after vector injection at P1. Mice injected through the round window membrane with PBS survived to at least P6, the last day evaluated. Of seven mice injected with AAV-CBA-mmGJB2, six died by P5. Mice injected with vector encoding a null mutant GJB2 (35delG) lived to at least P30. c. Overview of the experimental design. d. Identification of potential GREs with ATAC-seq. Ten cochleas from neonatal mice (P2-P8) were harvested and processed for ATAC-seq. Peaks representing open chromatin are shown for a ∼300 kb stretch of mouse genome near the Gjb2 locus. Peaks for a neuronal sample are shown at the top; sequence conservation among mammalian genomes is plotted at the bottom. e. Expression of a reporter driven by a ubiquitous promoter. An AAV9-PHP.B vector (AAV-CBA-eGFP) expressing eGFP (green) under a CBA promoter efficiently transduced inner hair cells (IHCs), outer hair cells (OHCs) (magenta), supporting cells, and other cells in mouse cochlea. IHCs and OHCs were identified by fluorescent phalloidin (magenta). f. Cells transduced with an AAV vector expressing the eGFP marker gene under the control of GREs (AAV-GRE-eGFP). Notably, no eGFP was observed in hair cells when expression was controlled by the GREs. g, h. Cells transduced with an AAV vectors express ing mmGJB2.HA ( g ) or hsGJB2.HA ( h ) under the control of regulatory elements. Scale bars: 10 μm ( e, f ), 30 µm ( g, h ).

Article Snippet: For immunofluorescence labeling, the following primary antibodies and secondary antibodies were used: mouse anti-MYO7A antibody (1:50) (#sc-74516, Santa Cruz), rabbit anti-HA C29F4 antibody (1:200) (#3724, Cell Signaling), rabbit anti-GJB2 antibody (1:50) (#71-0500, Fisher), goat anti-SOX2 antibody (1:50) (#AF2018, R&D Systems), rabbit anti-IBA1 antibody (1:200) (#019-19741, Wako Chemicals), donkey anti-rabbit IgG secondary antibody conjugated to Alexa Fluor 594 (1:200) (Invitrogen), donkey anti-goat IgG conjugated to Alexa Fluor 647 (1:200) (Invitrogen), donkey anti-mouse IgG conjugated to Alexa Fluor 488 (1:200) (Invitrogen), donkey anti-mouse IgG conjugated to Alexa Fluor 594 (1:200) (Invitrogen), and donkey anti-rabbit IgG conjugated to Alexa Fluor 488 (1:200) (Invitrogen).

Techniques: Virus, Plasmid Preparation, Injection, Membrane, Mutagenesis, Sequencing, Expressing, Transduction, Marker, Control

Journal: bioRxiv

Article Title: Cell-specific delivery of GJB2 restores auditory function in mouse models of DFNB1 deafness and mediates appropriate expression in NHP cochlea

doi: 10.1101/2024.12.24.630240

Figure Lengend Snippet:

Article Snippet: For immunofluorescence labeling, the following primary antibodies and secondary antibodies were used: mouse anti-MYO7A antibody (1:50) (#sc-74516, Santa Cruz), rabbit anti-HA C29F4 antibody (1:200) (#3724, Cell Signaling), rabbit anti-GJB2 antibody (1:50) (#71-0500, Fisher), goat anti-SOX2 antibody (1:50) (#AF2018, R&D Systems), rabbit anti-IBA1 antibody (1:200) (#019-19741, Wako Chemicals), donkey anti-rabbit IgG secondary antibody conjugated to Alexa Fluor 594 (1:200) (Invitrogen), donkey anti-goat IgG conjugated to Alexa Fluor 647 (1:200) (Invitrogen), donkey anti-mouse IgG conjugated to Alexa Fluor 488 (1:200) (Invitrogen), donkey anti-mouse IgG conjugated to Alexa Fluor 594 (1:200) (Invitrogen), and donkey anti-rabbit IgG conjugated to Alexa Fluor 488 (1:200) (Invitrogen).

Techniques: Expressing

Journal: bioRxiv

Article Title: Cell-specific delivery of GJB2 restores auditory function in mouse models of DFNB1 deafness and mediates appropriate expression in NHP cochlea

doi: 10.1101/2024.12.24.630240

Figure Lengend Snippet: a. Distribution of GJB2 ( magenta ) in Gjb2 Pfl/Pfl , Cre- control mouse cochlea ( left ) and Gjb2 Pfl/Pfl , Cre+ cochlea (right) at P6. In Gjb2 Pfl/Pfl , Cre- mice, GJB2 was observed in Deiters’ cells, Hensen’s cells, Claudius cells, pillar cells, border cells, the epithelium of the inner and outer sulcus and the root cells of spiral prominence; in fibrocytes in the lateral wall and spiral limbus; and in basal and intermediate cells in the stria vascularis. Hair cells, labeled with an antibody to parvalbumin (PV; yellow) did not express Gjb2 in control cochleas. In Gjb2 Pfl/Pfl , Cre+ conditional knockout mice at P6, anti-GJB2 labeling detected no signal hair cells or epithelial cells in the organ of Corti. b. At P6, scanning electron microscopy of hair cells showed normal bundle morphology in both Gjb2 Pfl/Pfl , Cre+ conditional knockout mice and Gjb2 Pfl/Pfl , Cre- controls. c. Distribution at P30 of GJB2 in Gjb2 Pfl/Pfl , Cre- ( left ) and Gjb2 Pfl/Pfl , Cre+ (right) mouse cochleas. Conditional knockout mice had developed degeneration of the organ of Corti by P30. d. Exogenous (vector-delivered) mmGJB2.HA expression in P30 Gjb2 Pfl/Pfl , Cre+ conditional knockout mouse cochlea. Hair cells did not express mmGJB2.HA. It appeared in the epithelium of the inner and outer sulcus and the root cells of the spiral prominence, fibrocytes type I, II and V of the lateral wall and fibrocytes of the spiral limbus and strial cells, and these cells survived to at least P30. Hair cells, missing at P30 in untreated Gjb2 Pfl/Pfl , Cre+ mice, were present in the vector-treated cochlea. LW - lateral wall, SL - spiral limbus, SGNs - spiral ganglion neurons. Scale bars: 30 μm ( a, c, d ), 2μm ( b ).

Article Snippet: For immunofluorescence labeling, the following primary antibodies and secondary antibodies were used: mouse anti-MYO7A antibody (1:50) (#sc-74516, Santa Cruz), rabbit anti-HA C29F4 antibody (1:200) (#3724, Cell Signaling), rabbit anti-GJB2 antibody (1:50) (#71-0500, Fisher), goat anti-SOX2 antibody (1:50) (#AF2018, R&D Systems), rabbit anti-IBA1 antibody (1:200) (#019-19741, Wako Chemicals), donkey anti-rabbit IgG secondary antibody conjugated to Alexa Fluor 594 (1:200) (Invitrogen), donkey anti-goat IgG conjugated to Alexa Fluor 647 (1:200) (Invitrogen), donkey anti-mouse IgG conjugated to Alexa Fluor 488 (1:200) (Invitrogen), donkey anti-mouse IgG conjugated to Alexa Fluor 594 (1:200) (Invitrogen), and donkey anti-rabbit IgG conjugated to Alexa Fluor 488 (1:200) (Invitrogen).

Techniques: Control, Labeling, Knock-Out, Electron Microscopy, Plasmid Preparation, Expressing

Journal: bioRxiv

Article Title: Cell-specific delivery of GJB2 restores auditory function in mouse models of DFNB1 deafness and mediates appropriate expression in NHP cochlea

doi: 10.1101/2024.12.24.630240

Figure Lengend Snippet: a. Average tone ABR thresholds as a function of frequency and click thresholds for P30 uninjected Gjb2 Pfl/Pfl , Cre- control mice (blue), uninjected Gjb2 Pfl/Pfl , Cre+ conditional knockout mice (black), and conditional knockout mice injected with AAV-GRE-mmGjb2.HA (green). b. Average DPOAEs thresholds at P30 for the same mice. Data are presented as mean ± SEM.

Article Snippet: For immunofluorescence labeling, the following primary antibodies and secondary antibodies were used: mouse anti-MYO7A antibody (1:50) (#sc-74516, Santa Cruz), rabbit anti-HA C29F4 antibody (1:200) (#3724, Cell Signaling), rabbit anti-GJB2 antibody (1:50) (#71-0500, Fisher), goat anti-SOX2 antibody (1:50) (#AF2018, R&D Systems), rabbit anti-IBA1 antibody (1:200) (#019-19741, Wako Chemicals), donkey anti-rabbit IgG secondary antibody conjugated to Alexa Fluor 594 (1:200) (Invitrogen), donkey anti-goat IgG conjugated to Alexa Fluor 647 (1:200) (Invitrogen), donkey anti-mouse IgG conjugated to Alexa Fluor 488 (1:200) (Invitrogen), donkey anti-mouse IgG conjugated to Alexa Fluor 594 (1:200) (Invitrogen), and donkey anti-rabbit IgG conjugated to Alexa Fluor 488 (1:200) (Invitrogen).

Techniques: Control, Knock-Out, Injection

Journal: bioRxiv

Article Title: Cell-specific delivery of GJB2 restores auditory function in mouse models of DFNB1 deafness and mediates appropriate expression in NHP cochlea

doi: 10.1101/2024.12.24.630240

Figure Lengend Snippet: a. Representative confocal microscopy images for GJB6 in Gjb2 Gjb6+ control cochleas ( left ) and Gjb2 Gjb6- mutants ( right ). Anti-GJB6 signal was detected in fibrocytes, Deiters’, Hensen’s, and Claudius cells, pillar cells and in both the inner and outer sulcus. No GJB6 immunoreactivity was observed in inner or outer hair cells. Gjb2 Gjb6- cochleas showed normal gross morphology of the organ of Corti, hair cells, but no GJB6 was detected immunoreactivity ( right ). b. Representative confocal microscopy images of side-by-side comparison of GJB2 expression in Gjb2 Gjb6- and Gjb2 Gjb6+ at P30. c. Representative confocal microscopy images of AAV- GRE-hsGJB2-treated Gjb2 Gjb6- mice show complete restoration of GJB2 expression to levels seen in Gjb2 Gjb6+ controls. Scale bars: 20 μm.

Article Snippet: For immunofluorescence labeling, the following primary antibodies and secondary antibodies were used: mouse anti-MYO7A antibody (1:50) (#sc-74516, Santa Cruz), rabbit anti-HA C29F4 antibody (1:200) (#3724, Cell Signaling), rabbit anti-GJB2 antibody (1:50) (#71-0500, Fisher), goat anti-SOX2 antibody (1:50) (#AF2018, R&D Systems), rabbit anti-IBA1 antibody (1:200) (#019-19741, Wako Chemicals), donkey anti-rabbit IgG secondary antibody conjugated to Alexa Fluor 594 (1:200) (Invitrogen), donkey anti-goat IgG conjugated to Alexa Fluor 647 (1:200) (Invitrogen), donkey anti-mouse IgG conjugated to Alexa Fluor 488 (1:200) (Invitrogen), donkey anti-mouse IgG conjugated to Alexa Fluor 594 (1:200) (Invitrogen), and donkey anti-rabbit IgG conjugated to Alexa Fluor 488 (1:200) (Invitrogen).

Techniques: Confocal Microscopy, Control, Comparison, Expressing

Journal: bioRxiv

Article Title: Cell-specific delivery of GJB2 restores auditory function in mouse models of DFNB1 deafness and mediates appropriate expression in NHP cochlea

doi: 10.1101/2024.12.24.630240

Figure Lengend Snippet: HA- tagged GJB2 expression 30 days post-injection. Despite strong labeling for hsGJB2.HA, multiple abnormalities were detected: reduced scala media volume, thickened tectorial membrane, enlarged inner sulcus cells, absent or reduced tunnel of Corti, and loss of OHCs.

Article Snippet: For immunofluorescence labeling, the following primary antibodies and secondary antibodies were used: mouse anti-MYO7A antibody (1:50) (#sc-74516, Santa Cruz), rabbit anti-HA C29F4 antibody (1:200) (#3724, Cell Signaling), rabbit anti-GJB2 antibody (1:50) (#71-0500, Fisher), goat anti-SOX2 antibody (1:50) (#AF2018, R&D Systems), rabbit anti-IBA1 antibody (1:200) (#019-19741, Wako Chemicals), donkey anti-rabbit IgG secondary antibody conjugated to Alexa Fluor 594 (1:200) (Invitrogen), donkey anti-goat IgG conjugated to Alexa Fluor 647 (1:200) (Invitrogen), donkey anti-mouse IgG conjugated to Alexa Fluor 488 (1:200) (Invitrogen), donkey anti-mouse IgG conjugated to Alexa Fluor 594 (1:200) (Invitrogen), and donkey anti-rabbit IgG conjugated to Alexa Fluor 488 (1:200) (Invitrogen).

Techniques: Expressing, Injection, Labeling, Membrane

Journal: bioRxiv

Article Title: Cell-specific delivery of GJB2 restores auditory function in mouse models of DFNB1 deafness and mediates appropriate expression in NHP cochlea

doi: 10.1101/2024.12.24.630240

Figure Lengend Snippet: a. Untreated Gjb2 Gjb6+ mice displayed normal hearing, while untreated Gjb2 Gjb6- mice were profoundly deaf by P30. Hearing function was also evaluated in Gjb2 Gjb6- mice treated with AAV-GRE-hsGJB2.HA. ABRs were measured in response to broadband clicks and tone bursts (left), alongside DPOAEs (right). Gjb2 Gjb6- mice injected with AAV-GRE-hsGJB2.HA showed no improvement in hearing. b. Control mice injected with AAV- GRE-hsGJB2.HA displayed ABR responses comparable to untreated control mice. However, their DPOAE levels were slightly elevated. c. At P30, Gjb2 Gjb6- mice treated with either a high dose (1.4×10¹¹ VGC) or low dose (7.0×10¹⁰ VGC) of AAV-GRE-hsGJB2 demonstrated full recovery of click-evoked and tone burst ABR responses, matching the levels observed in untreated control mice. d. Hearing recovery persisted through P60, with treated mice maintaining ABR responses equivalent to those of normal hearing controls. At P30 in e and P60 in d , the amplitude of ABR wave 1 (from P1 to N1) at 16 kHz in low-dose and high- dose treated mice. g, h. DPOAE measurements in Gjb2 Gjb6- mice treated with AAV-GRE-hsGJB2. At P30 ( g ), DPOAE measurements demonstrated robust rescue in mice treated at P1 with either the high or low dose of AAV-GRE-hsGJB2. This rescue persisted at P60 ( h ), with DPOAE levels comparable to normal hearing mice.

Article Snippet: For immunofluorescence labeling, the following primary antibodies and secondary antibodies were used: mouse anti-MYO7A antibody (1:50) (#sc-74516, Santa Cruz), rabbit anti-HA C29F4 antibody (1:200) (#3724, Cell Signaling), rabbit anti-GJB2 antibody (1:50) (#71-0500, Fisher), goat anti-SOX2 antibody (1:50) (#AF2018, R&D Systems), rabbit anti-IBA1 antibody (1:200) (#019-19741, Wako Chemicals), donkey anti-rabbit IgG secondary antibody conjugated to Alexa Fluor 594 (1:200) (Invitrogen), donkey anti-goat IgG conjugated to Alexa Fluor 647 (1:200) (Invitrogen), donkey anti-mouse IgG conjugated to Alexa Fluor 488 (1:200) (Invitrogen), donkey anti-mouse IgG conjugated to Alexa Fluor 594 (1:200) (Invitrogen), and donkey anti-rabbit IgG conjugated to Alexa Fluor 488 (1:200) (Invitrogen).

Techniques: Injection, Control

Journal: bioRxiv

Article Title: Cell-specific delivery of GJB2 restores auditory function in mouse models of DFNB1 deafness and mediates appropriate expression in NHP cochlea

doi: 10.1101/2024.12.24.630240

Figure Lengend Snippet: a. Overview of the experimental design. b. Robust eGFP expression was observed in an NHP cochlea injected with AAV-CBA-eGFP. eGFP signal (green) was detected in hair cells, Deiters’ cells, Hensen’s cells, Claudius cells, pillar cells, border cells, the epithelium of the inner and outer sulcus and the root cells of the spiral prominence, in fibrocytes type I-V in the lateral wall and in the cells of the stria vascularis, in spiral limbus fibrocytes and in interdental cells. DAPI labeled nuclei (blue). c. Distribution of endogenous GJB2 (yellow) in an NHP cochlea. GJB2 signal was detected in the following supporting cells (magenta): Deiters’ cells (DCs), Hensen’s cells, Claudius cells, pillar cells (PCs), and border cells. The GJB2 network was seen in the epithelium of the inner and outer sulcus and the root cells of spiral prominence (SP), in fibrocytes type I, II and V in the lateral wall and in basal and intermediate cells in the stria vascularis (SV). The spiral limbus fibrocytes also contained many GJB2 puncta. Neither outer hair cells (OHCs) nor inner hair cells (IHCs) (red) expressed GJB2. DAPI labeled nuclei (blue). d. Exogenous (vector-delivered) GJB2.HA expression in an NHP cochlea. The HA tag was detected in SOX2-positive cells (magenta), such as in Deiters’ cells, Hensen’s cells, Claudius cells and border cells. Little or no HA labeling was seen in pillar cells. IHCs and OHCs (yellow) do not express GJB2.HA. The GJB2-HA network was seen in the epithelium of the inner and outer sulcus and the root cells of SP, in fibrocytes type I, II and V of the lateral wall and fibrocytes of the spiral limbus. The strial cells expressed exogenous GJB2.HA at a significantly low level. DAPI labeled nuclei (blue). Scale bars are 500 µm.

Article Snippet: For immunofluorescence labeling, the following primary antibodies and secondary antibodies were used: mouse anti-MYO7A antibody (1:50) (#sc-74516, Santa Cruz), rabbit anti-HA C29F4 antibody (1:200) (#3724, Cell Signaling), rabbit anti-GJB2 antibody (1:50) (#71-0500, Fisher), goat anti-SOX2 antibody (1:50) (#AF2018, R&D Systems), rabbit anti-IBA1 antibody (1:200) (#019-19741, Wako Chemicals), donkey anti-rabbit IgG secondary antibody conjugated to Alexa Fluor 594 (1:200) (Invitrogen), donkey anti-goat IgG conjugated to Alexa Fluor 647 (1:200) (Invitrogen), donkey anti-mouse IgG conjugated to Alexa Fluor 488 (1:200) (Invitrogen), donkey anti-mouse IgG conjugated to Alexa Fluor 594 (1:200) (Invitrogen), and donkey anti-rabbit IgG conjugated to Alexa Fluor 488 (1:200) (Invitrogen).

Techniques: Expressing, Injection, Labeling, Plasmid Preparation

Journal: bioRxiv

Article Title: Cell-specific delivery of GJB2 restores auditory function in mouse models of DFNB1 deafness and mediates appropriate expression in NHP cochlea

doi: 10.1101/2024.12.24.630240

Figure Lengend Snippet: a. Vehicle-injected cochlea. Anti-SOX2 labeling (magenta), anti-MYO7A labeling (yellow) and DAPI-labeled nuclei (blue) were detected. b. AAV- GRE-hsGJB2.HA-injected cochlea. Robust anti-HA labeling (red) was detected in the same locations as endogenous GJB2, analysis of cochlear turns showed no gradient in GJB2.HA expression between apex, middle and base. Scale bars = 1 mm.

Article Snippet: For immunofluorescence labeling, the following primary antibodies and secondary antibodies were used: mouse anti-MYO7A antibody (1:50) (#sc-74516, Santa Cruz), rabbit anti-HA C29F4 antibody (1:200) (#3724, Cell Signaling), rabbit anti-GJB2 antibody (1:50) (#71-0500, Fisher), goat anti-SOX2 antibody (1:50) (#AF2018, R&D Systems), rabbit anti-IBA1 antibody (1:200) (#019-19741, Wako Chemicals), donkey anti-rabbit IgG secondary antibody conjugated to Alexa Fluor 594 (1:200) (Invitrogen), donkey anti-goat IgG conjugated to Alexa Fluor 647 (1:200) (Invitrogen), donkey anti-mouse IgG conjugated to Alexa Fluor 488 (1:200) (Invitrogen), donkey anti-mouse IgG conjugated to Alexa Fluor 594 (1:200) (Invitrogen), and donkey anti-rabbit IgG conjugated to Alexa Fluor 488 (1:200) (Invitrogen).

Techniques: Injection, Labeling, Expressing

Journal: Cell reports

Article Title: Hair follicles modulate skin barrier function

doi: 10.1016/j.celrep.2024.114347

Figure Lengend Snippet: (A) LacZ staining, as a readout for Abca12 promoter activity, in K14-Cre ERT ;Abca12-KO/cKO (KAL) or Lrig1-Cre ERT2 ;Abca12-KO/cKO (LAL) mice. Controls are Abca12-KO/cKO mice lacking Cre. (B) IHC for Abca12 in Lrig1-Cre ERT2 ;Abca12-c/c;ROSA-YFP (LAY) mice 2 weeks post-TAM. Control mice are similar to LAY mutants but possess one wild-type copy of Abca12 ( Abca12-c/+ ). Right images are magnified, single-channel views showing non-cell-autonomous upregulation of Abca12 (red) in LAY mutant skin that does not overlap with YFP+ uHF cells (green). (C) Same as (B) but with IHC for Ki67 (red) 10 weeks post-TAM. (D) Quantitation of proliferation in YFP-negative, basal IFE cells. (E) Volcano plot showing upregulated (red) and downregulated (blue) differentially expressed genes (DEGs) in KA mice compared to control Abca12-c/c mice lacking Cre. (F) KEGG enrichment analysis of DEGs. (G) Venn diagrams showing overlap of DEGs in KA mice, patients with HI, and Abca12 -mutant embryonic mice. Overlapping genes from the 3 datasets are depicted in (E). (H) Same as (B) but with IHC for Sprr2d (red, left) or Gjb2 (red, right) 2 weeks post-TAM. For (D): * p < 0.05, ** p < 0.01, and *** p < 0.001 by unpaired t test, comparing only samples from the same time point. n ≥ 4 mice per genotype per time point. Error bars indicate mean ± SD. Scale bar, 50 μm. See also and .

Article Snippet: Rabbit anti-Gjb2 , Fisher Scientific , Cat # 71–0500.

Techniques: Staining, Activity Assay, Control, Mutagenesis, Quantitation Assay

Journal: Cell reports

Article Title: Hair follicles modulate skin barrier function

doi: 10.1016/j.celrep.2024.114347

Figure Lengend Snippet: (A) IHC for YFP (green) confirming recombination in K79+ uHF cells in K79-Cre;ROSA-YFP mice at P12. (B and C) Gross photos of P7 pups of the indicated genotypes. Only K79-Cre;Abca12-LacZ/c pups exhibited severe skin thickening and flakiness. (D) Quantitation of epidermal thickness for P7 pups of the indicated genotypes. Each data point represents a single mouse. (E) IHC for Abca12 (red) confirming that the protein was not ablated from the IFE of K79-Cre;Abca12-LacZ/c skin at P7. Bottom images are single-channel views of Abca12 staining. (F) IHC for Sprr2d (red) in K79-Cre;Abca12-LacZ/c or control skin at P7. (G) Same as (F) but with staining for Gjb2 (red). (H) Left image, gross photos of two P6 mutant pups, both of genotype K79-Cre;Abca12-LacZ/c with ROSA-YFP . Right image, IHC showing YFP expression (green) in developing hair canals after Cre-mediated recombination and thickened epidermis (red). Asterisk, hyperkeratotic material obstructing hair canals, visible by bright-field overlay. Dotted line denotes the top of the epidermis. See for IHC of littermate control skin. For (D): * p < 0.05 by one-way ANOVA with post hoc Tukey test, comparing K79-Cre;Abca12-LacZ/c pups against Abca12-LacZ/c or K79-Cre;Abca12-c/c pups. n ≥ 6 mice per genotype. Error bars indicate mean ± SD. Scale bar, 50 μm. See also .

Article Snippet: Rabbit anti-Gjb2 , Fisher Scientific , Cat # 71–0500.

Techniques: Quantitation Assay, Staining, Control, Mutagenesis, Expressing

Journal: Cell reports

Article Title: Hair follicles modulate skin barrier function

doi: 10.1016/j.celrep.2024.114347

Figure Lengend Snippet:

Article Snippet: Rabbit anti-Gjb2 , Fisher Scientific , Cat # 71–0500.

Techniques: Control, Recombinant, Electron Microscopy, RNAscope, RNA Sequencing Assay, Mutagenesis, In Situ, Software, Clone Assay

Journal: Biomedicines

Article Title: The Interplay of Cx26, Cx32, Cx37, Cx40, Cx43, Cx45, and Panx1 in Inner-Ear Development of Yotari (dab1−/−) Mice and Humans

doi: 10.3390/biomedicines10030589

Figure Lengend Snippet: List of primary and secondary antibodies.

Article Snippet: Primary , Cx26, GJB2 , Rabbit , 1:50 , Cusabio (Wuhan, China) , CSBPA009452LA01HU.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Connexin Expression Is Altered in Liver Development of Yotari ( dab1 -/- ) Mice

doi: 10.3390/ijms221910712

Figure Lengend Snippet: Immunofluorescence staining of connexins (Cxs): Cx26 and Cx32 in wild type and yotari mouse liver at gestation days E13.5 and E15.5. Immunoexpression of Cx26, 4′,6-diamidino-2- phenylindole dihydrochloride (DAPI) staining and merged Cx26 and DAPI at E13.5 and E15.5 in wild type ( a ) and yotari ( b ). Immunoexpression of Cx32, DAPI staining and merged Cx32 and DAPI at E13.5 and E15.5 in wild type ( c ) and yotari ( d ). * Autofluorescence of erythrocytes not to be mistaken with positive immunofluorescence staining of connexins. The scale bar is 8µm, refers to all images.

Article Snippet: Primary , Cx26, GJB2 , CSB-PA009452LA01HU , Rabbit , 1:50 , Cusabio (Wuhan, China).

Techniques: Immunofluorescence, Staining

Journal: International Journal of Molecular Sciences

Article Title: Connexin Expression Is Altered in Liver Development of Yotari ( dab1 -/- ) Mice

doi: 10.3390/ijms221910712

Figure Lengend Snippet: The area percentages of connexins (Cxs): Cx26, Cx32, Cx37, Cx40, Cx43, Cx45, pannexin1 (Panx1) and apoptosis-inducing factor (AIF) in wild type and yotari livers at gestation days E13.5 and E15.5. Data are presented as the mean ± SD (vertical line). Significant differences were indicated by * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. One-way ANOVA followed by Tukey’s multiple comparisons test.

Article Snippet: Primary , Cx26, GJB2 , CSB-PA009452LA01HU , Rabbit , 1:50 , Cusabio (Wuhan, China).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Connexin Expression Is Altered in Liver Development of Yotari ( dab1 -/- ) Mice

doi: 10.3390/ijms221910712

Figure Lengend Snippet: Primary and secondary antibodies used in the study.

Article Snippet: Primary , Cx26, GJB2 , CSB-PA009452LA01HU , Rabbit , 1:50 , Cusabio (Wuhan, China).

Techniques: Recombinant

Journal: bioRxiv

Article Title: Single cell transcriptomics of human epidermis reveals basal stem cell transition states

doi: 10.1101/784579

Figure Lengend Snippet: A) Heatmap showing top 300 differentially expressed genes per cluster. Dotted lines outline differentially expressed genes. Select marker genes are shown on the right. B) Violin plots of relative expression of marker genes split by cell cohorts and color-coded by cell community as in A. C) Feature plots showing expression of select basal and granular cell marker genes. D) Immunostaining of KRT14 (red), KRT10 (green), and DAPI (blue) in human neonatal skin. Scale bar 100µm. E-J) Immunostaining of differentially expressed proteins (red) from the (E) BAS-III cluster (ASS1 and COL17A1), (F) BAS-I cluster (PTTG1 and CDC20), (G) BAS-III/IV clusters (KRT19 and GJB2), (H) BAS-II cluster (RRM2 and PCLAF), (I) GRN cluster (SPINK5 and CALML5), and (J) the MEL (MLANA) and LAN (CD74) clusters in human neonatal skin. COL7A1 (green) and DAPI (blue) are co-stained. Dotted lines denote position of basement membrane where COL7A1 staining is absent. Scale bar 100µm.

Article Snippet: The following antibodies were used: chicken anti-KRT14 (1:500; BioLegend; SIG-3476), mouse anti-KRT10 (1:500; Dako; M7002), rabbit anti-KI67 (1:500; Abcam; ab15580), rabbit anti-PTTG1 (1:100; Sigma-Aldrich; HPA008890), mouse anti-ASS1 (1:100; Santa Cruz; sc-365475), rabbit anti-COL17A1 (1:100; One World Labs; ap9099c), rabbit anti-COL7A1 (1:500; abcam; ab93350), mouse anti-COL7A1 (1:100; Santa Cruz; sc-33710), rabbit anti-GJB2 (1:250; ThermoFisher; 51-2800), rabbit anti-KRT19 (1:250; Cell signaling; 13092), mouse anti-SPINK5 (1:100; Santa Cruz; sc-137109), mouse anti-CALML5 (1:100; Santa Cruz; sc-393637), mouse anti-CDC20 (1:100; Santa Cruz; sc-13162), mouse anti-RRM2 (1:100; Santa Cruz; sc-398294, sc-376973), mouse anti-PCLAF (1:100; Santa Cruz; sc-390515), mouse anti-MLANA (1:100; Santa Cruz; sc-20032), and mouse anti-CD74 (1:100; Santa Cruz; sc-6262).

Techniques: Marker, Expressing, Immunostaining, Staining