Journal: The Journal of Clinical Investigation

Article Title: In vivo visualization and molecular targeting of the cardiac conduction system

doi: 10.1172/JCI156955

Figure Lengend Snippet: ( A ) Antibody-dye conjugate (mCntn2-800) consists of a near-infrared (NIR) dye conjugated to an antibody against the CCS-specific surface marker Cntn2 (contactin 2). ( B ) Experimental work flow. ( C ) Whole-body biodistribution of other tissue types, showing expected clearance within the liver, bladder, and kidneys and notable absence from the brain. ( D ) Whole mouse heart ( n = 3) from a wild-type (WT) mouse injected 3 days prior with mCntn2-800, in posterior-anterior (PA) and right lateral (RL) views. Atria are outlined in white and cardiac chambers are listed. LA, left atrium; LV, left ventricle; RA, right atrium; RV, right ventricle. Top: Brightfield. Bottom: NIR signal demonstrating labeling of the CCS (blue→red = lowest→highest signal). Mean signal to background ratio (SBR) is indicated. ( E ) Measured intervals (in ms) from sedated surface electrocardiograms (ECGs) including PR, QRS, QTc, and RR in WT mice prior to (day 0 = baseline, n = 12) and daily (day 1 n = 3, day 2 n = 9; after injection) following a single tail vein injection of mCntn2-800. Intervals (mean ± SD) on a given day after injection were compared to each mouse’s preinjection control baseline (day 0) using 1-way ANOVA with Tukey’s post hoc test. ( F – H ) Heart sections from a WT mouse injected 2 days prior with mCntn2-800. CCS components labeled with mCntn2-800 (purple) and costained with antibodies targeting known markers of the CCS, including anti-Hcn4 (red, SAN and His) or anti-Cx40 (green, PF). DAPI (blue, nuclei). LBB/RBB, left and right bundle branches; His, His bundle; PF, Purkinje fibers; SAN, sinoatrial node; VM, ventricular myocardium. Scale bars: 10 mm ( C ), 5 mm ( D ), and 100 μm ( F – H ).

Article Snippet: Described antibody-dye conjugates consist of commercially acquired anti-Cntn2 goat polyclonal antibody (AF4439) and anti-NPTN goat polyclonal antibody (AF5360) (both R&D Systems) that have been covalently conjugated to a benign, NIR dye (IRDye800CW NHS ester; LI-COR Biosciences, 929-70020) using company specifications.

Techniques: Marker, Injection, Labeling

Journal: The Journal of Clinical Investigation

Article Title: In vivo visualization and molecular targeting of the cardiac conduction system

doi: 10.1172/JCI156955

Figure Lengend Snippet: ( A ) Experimental workflow: wild-type mice ( n = 3) received a tail vein injection of mCntn2-800 and after 48 hours were sedated and received a sternotomy and cardiac incisions including a right atriotomy and right ventriculotomy to simulate a surgical scenario. Live imaging of the heart with a FLARE Intraoperative Near-Infrared (NIR) Fluorescence Imaging System. ( B and C ) Heart with visible sinoatrial node (SAN), atrioventricular node/His bundle (AVN/His), and Purkinje fiber (PF) network. Left: Color image of ex vivo heart. Right: mCntn2-800 NIR signal (green). Middle: Merged image of color image and NIR (green) signal. ( D ) Heart sections from the same heart, demonstrating mCntn2-800 signal (cyan) labeling the SAN, as costained with anti-Cntn2 (red) immunostaining. ( E ) Magnified region in SAN indicated by the white box in D . Right atrial (RA) tissue labeled with anti-Cx43 (purple). DAPI (blue, nuclei). IVS, interventricular septum; RV, right ventricle; SAN, sinoatrial node; SVC, superior vena cava. Scale bars: 5 mm ( B and C ), 300 μm ( D ), and 100 μm ( E ).

Article Snippet: Described antibody-dye conjugates consist of commercially acquired anti-Cntn2 goat polyclonal antibody (AF4439) and anti-NPTN goat polyclonal antibody (AF5360) (both R&D Systems) that have been covalently conjugated to a benign, NIR dye (IRDye800CW NHS ester; LI-COR Biosciences, 929-70020) using company specifications.

Techniques: Injection, Imaging, Fluorescence, Ex Vivo, Labeling, Immunostaining

Journal: The Journal of Clinical Investigation

Article Title: In vivo visualization and molecular targeting of the cardiac conduction system

doi: 10.1172/JCI156955

Figure Lengend Snippet: ( A ) Human anti-CNTN2 Fab antibody was biotinylated and conjugated to streptavidin-linked saporin, a cell toxin (hCNTN2-Sap). ( B ) Wild-type mice received a single tail vein injection of either hCNTN2-Sap (100 μg) ( n = 6) or Control-Sap (100 μg nonspecific human IgG similarly conjugated to saporin) ( n = 6). Mice received electrocardiograms (ECGs) on day 0 (baseline) and daily following injection with control or hCNTN2-Sap. On day 2, hearts were harvested, fixed, and immunostained. ( C ) Representative ECG tracings ( n = 6 per condition). ( D ) By day 2, mice injected with hCNTN2-Sap demonstrated marked conduction abnormalities, including prolonged PR, QRS, and RR intervals as compared with mice injected with Control-Sap (mean intervals in ms ± SD). Gray bar, QTc interval corrected for QRS intervals. Statistical analyses using 2-way ANOVA with Tukey’s post hoc test. ( E ) Consistent with targeted cell death, immunofluorescence of the CCS (red) showed subtotal loss of CCS cells as shown within the His bundle (His), right and left bundle branches (RBB/LBB) as stained by anti-Cntn2. His, His bundle; IVS, interventricular septum; LBB, left bundle branch; RBB, right bundle branch. Scale bar: 50 μm.

Article Snippet: Described antibody-dye conjugates consist of commercially acquired anti-Cntn2 goat polyclonal antibody (AF4439) and anti-NPTN goat polyclonal antibody (AF5360) (both R&D Systems) that have been covalently conjugated to a benign, NIR dye (IRDye800CW NHS ester; LI-COR Biosciences, 929-70020) using company specifications.

Techniques: Injection, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Nhlh1 and Nhlh2, a global transcriptional mechanism regulating commissural axon projection via activating Robo3

doi: 10.1101/2022.09.23.509112

Figure Lengend Snippet: Lack of ventral commissures in the spinal cord, hindbrain and midbrain in Nhlh1 and Nhlh2 double mutant. Coronal sections from the spinal cord (A), caudal hindbrain (B), rostral hindbrain (C), and midbrain (D) at E11.5 were subjected to Tag1 and NF double IHC. The Tag1 signals label the commissural axons and the NF signals show the general axonal patterns. The DAPI counterstaining indicate the overall cytoarchitecture. Comparisons were made between the control genotype ( Nhlh1 +/+ Nhlh2 +/m ) (n = 3) and the double mutant ( Nhlh1 m/m Nhlh2 m/m ) (n = 3). The Tag1 and NF merged images in the bottom panel are high magnification images of the ventral commissure regions. Filled arrows show the ventral commissures and the hollow arrows show the lack of ventral commissures. Scale bars: 200 μm in low magnification images in (A), (B), (C) & (D); 200 μm in high magnification images in (A), (B), (C) & (D).

Article Snippet: The primary antibodies used were: rabbit anti-Barhl1 polyclonal antibody (Atlas Antibodies, HPA004809, Sigma, 1:500), goat anti-Robo3 polyclonal antibody (R&D Systems, AF3076, 1:200), goat anti-Tag1 polyclonal antibody (R&D Systems, AF4439, 1:500), mouse anti-Neurofilament-160KD monoclonal antibody (clone: RMO-270, Zymed, 13-0700, 1:500), rat anti-L1CAM monoclonal antibody (clone 324, Chemicon, MAB5272, 1:400), goat anti-DCC polyclonal antibody (Santa Cruz Biotechnology, sc-6535, 1:200), rabbit anti-FoxP2 polyclonal antibody (abcam, ab16046, 1:1000), mouse anti-Brn3a monoclonal antibody (clone: 5A3.2, Millipore, MAB1585, 1;200), chick anti-GFP polyclonal antibody (abcam, ab13970, 1:1500).

Techniques: Mutagenesis

Journal: bioRxiv

Article Title: Nhlh1 and Nhlh2, a global transcriptional mechanism regulating commissural axon projection via activating Robo3

doi: 10.1101/2022.09.23.509112

Figure Lengend Snippet: Commissure formation does not appear to be affected in Nhlh1 and Nhlh2 single mutant. Commissural axons were labelled by Tag1 IHC, and the overall axonal patterns were labelled by NF IHC. Ventral commissure appears comparable between the control genotype of Nhlh1 +/+ Nhlh2 +/m (n = 2), and the mutant genotypes of Nhlh1 +/m Nhlh2 m/m (n = 2) and Nhlh1 m/m Nhlh2 +/m (n = 2), in the spinal cord (A), caudal hindbrain (B), rostral hindbrain (C), and midbrain (D). Higher magnification images of the commissural region across the ventral midline are shown in the bottom panels. Scale bars: 100μm in (A); 200 μm in (B), (C) & (D).

Article Snippet: The primary antibodies used were: rabbit anti-Barhl1 polyclonal antibody (Atlas Antibodies, HPA004809, Sigma, 1:500), goat anti-Robo3 polyclonal antibody (R&D Systems, AF3076, 1:200), goat anti-Tag1 polyclonal antibody (R&D Systems, AF4439, 1:500), mouse anti-Neurofilament-160KD monoclonal antibody (clone: RMO-270, Zymed, 13-0700, 1:500), rat anti-L1CAM monoclonal antibody (clone 324, Chemicon, MAB5272, 1:400), goat anti-DCC polyclonal antibody (Santa Cruz Biotechnology, sc-6535, 1:200), rabbit anti-FoxP2 polyclonal antibody (abcam, ab16046, 1:1000), mouse anti-Brn3a monoclonal antibody (clone: 5A3.2, Millipore, MAB1585, 1;200), chick anti-GFP polyclonal antibody (abcam, ab13970, 1:1500).

Techniques: Mutagenesis

Journal: bioRxiv

Article Title: Nhlh1 and Nhlh2, a global transcriptional mechanism regulating commissural axon projection via activating Robo3

doi: 10.1101/2022.09.23.509112

Figure Lengend Snippet: Commissure-less phenotype in the Nhlh1 and Nhlh2 double mutant persisted into later developmental stages. Commissure formation was analyzed either by Tag1 or NF IHC. Lack of commissure formation continued to be observed in E13.5 spinal cord (A) (n = 2 for each genotype), E16.5 spinal cord (B) (n = 2 for each genotype), E13.5 hindbrain (C) (n = 2 for each genotype) and E13.5 midbrain (D) (n = 2 for each genotype). Scale bars: 200 μm in (A); 100 μm in (B); 400 μm in (C) & (D).

Article Snippet: The primary antibodies used were: rabbit anti-Barhl1 polyclonal antibody (Atlas Antibodies, HPA004809, Sigma, 1:500), goat anti-Robo3 polyclonal antibody (R&D Systems, AF3076, 1:200), goat anti-Tag1 polyclonal antibody (R&D Systems, AF4439, 1:500), mouse anti-Neurofilament-160KD monoclonal antibody (clone: RMO-270, Zymed, 13-0700, 1:500), rat anti-L1CAM monoclonal antibody (clone 324, Chemicon, MAB5272, 1:400), goat anti-DCC polyclonal antibody (Santa Cruz Biotechnology, sc-6535, 1:200), rabbit anti-FoxP2 polyclonal antibody (abcam, ab16046, 1:1000), mouse anti-Brn3a monoclonal antibody (clone: 5A3.2, Millipore, MAB1585, 1;200), chick anti-GFP polyclonal antibody (abcam, ab13970, 1:1500).

Techniques: Mutagenesis

Journal: The Journal of Neuroscience

Article Title: Genome Stability by DNA Polymerase β in Neural Progenitors Contributes to Neuronal Differentiation in Cortical Development

doi: 10.1523/JNEUROSCI.0665-17.2017

Figure Lengend Snippet: Polβ deficiency affects corticofugal axonal growth. Cortical layer IV neurons were labeled with EGFP by in utero electroporation at E13.5 and analyzed at E16.5. Immunohistochemical analyses were performed with anti-GFP antibody (A, C, D, F) in Polβfl/fl (A–C) and Emx1-Cre/Polβfl/fl mice (D–F). Nuclei were stained with DAPI (B, C, E, F). Scale bar, 500 μm. Frames indicate electroporated area. Asterisks indicate the center of electroporated area. Arrow and arrowhead indicate tips of corticofugal axons and commissural axons, respectively. G, Quantitative analysis of the length of axons was calculated from the center of each electroporated area to tips of axons in Polβfl/fl and Emx1-Cre/Polβfl/fl mice. **p < 0.01 (Mann–Whitney U test). Values indicate the mean ± SEM from 11 sections of 4 Polβfl/fl brains or 16 sections of 5 Emx1-Cre/Polβfl/fl brains. Immunohistochemistry was performed with anti-TAG-1 antibody (H–O) in E16.5 Polβfl/fl (H–K) and Emx1-Cre/Polβfl/fl (L–O) forebrain. Nuclei were stained with DAPI (I, K, M, O). Scale bar, 500 μm. J, K, N, O, Magnified images of the boxed area in I and M, respectively. Scale bar, 300 μm. IC, Internal capsule. Arrows indicate TAG-1-positive axons. P, Relative gene expression of Gap43, Apc, Tubb6, and Rplp1 was examined by qRT-PCR in E14.5 Polβfl/fl and Emx1-Cre/Polβfl/fl cortices. Gap43 and Apc are known to relate to axonal growth. Tubb6 and Rplp1 are ubiquitously expressed genes. Significant difference from Polβfl/fl cortices: **p < 0.01 (Student's t test).

Article Snippet: The sections were incubated overnight at 4°C with the following antibodies in buffer G or buffer D: rabbit polyclonal anticleaved caspase-3 (Asp175) (9661S, Cell Signaling Technology) at 1:250, mouse monoclonal anti–neuron-specific β-III tubulin (Tuj1) (MAB1195, R&D Systems) at 1:1000, goat polyclonal anti-Nurr1 (AF2156, R&D Systems) at 1:50, rabbit polyclonal anti-Tbr1 (ab31940, Abcam) at 1:500, rat monoclonal anti-Ctip2 (ab18465, Abcam) at 1:800, rabbit polyclonal anti-CDP (Cux1) (sc-13024, Santa Cruz Biotechnology) at 1:200, rat monoclonal anti-GFP (04404-84, Nacalai Tesque) at 1:1000, goat polyclonal anti-Contaction-2 (TAG-1) (AF4439, R&D Systems) at 1:200, rabbit polyclonal anti-histone H2AX phosphor Ser139 (γH2AX) (39117, Active Motif) at 1:200, rabbit monoclonal anti-Ki67 (RM-9106-S0, Thermo Scientific) at 1:200, or rabbit polyclonal anti-53BP1 (GTX102595, GeneTex) at 1:100.

Techniques: Labeling, In Utero, Electroporation, Immunohistochemical staining, Staining, MANN-WHITNEY, Immunohistochemistry, Expressing, Quantitative RT-PCR