Journal: Journal of Cardiovascular Development and Disease

Article Title: Nkx2-5 Loss of Function in the His-Purkinje System Hampers Its Maturation and Leads to Mechanical Dysfunction

doi: 10.3390/jcdd10050194

Figure Lengend Snippet: Condition deletion of Nkx2-5 in the ventricular conduction system. ( A ) Nkx2-5 immunofluorescence on transversal sections of P10 hearts from control (CTL) and Nkx2-5 ∆ VCS mutant mice. High magnification of the squares is represented below. IVS: interventricular septum; pm: papillary muscles. Scale bars = 100 µm; high-magnification image bar = 25 µm. ( B ) Co-immunofluorescence of Nkx2-5 and Cx40 or CNTN2 and Cx40 -RFP on serial sections of P10 Nkx2-5 ∆ VCS mutant hearts. Pecam1 and Dapi staining were used to delineate cardiac contours and nuclei. Scale bar = 50 µm.

Article Snippet: Antibodies used in this study are specific to Nkx2-5 (Sc8697 Santa-Cruz, Dallas, TX, USA), GFP (AbD Serotec, Purchheim, Germany), RFP (Rockland, Pottstown, PA, USA), Contactin-2 (AF1714 R&D system, Minneapolis, MN, USA), Pecam-1 (MEC13.3-BD Pharmingen, San Jose, CA, USA), HCN4 (Millipore, Burlington, MA, USA), ETV-1 (Abcam, Cambridge, U.K.), Troponin I (Abcam) and WGA-Cy3 (Sigma, St. Louis, MO, USA).

Techniques: Immunofluorescence, Mutagenesis, Staining

Journal: Journal of Cardiovascular Development and Disease

Article Title: Nkx2-5 Loss of Function in the His-Purkinje System Hampers Its Maturation and Leads to Mechanical Dysfunction

doi: 10.3390/jcdd10050194

Figure Lengend Snippet: Nkx2-5 VCS-conditional deletion mice present PF hypoplasia by cell dropout. ( A ) Whole-mount immunofluorescence with Contactin-2 on opened LV from 3-month-old control and Nkx2-5 ∆ VCS hearts. Scale bar = 1 mm. ( B ) Genetic tracing of ventricular Cx40 -positive cells after Cre induction at E18.5 in control and Nkx2-5 ∆ VCS mice showing the distribution of YFP+ cells in the PF network indicated by a co-immunofluorescence with CNTN2 at P20. High magnifications of peripheral PFs are presented in inserts. Scale bar = 0.5 mm. Below are sections from genetic tracing experiments stained with YFP and CNTN2 antibodies. Scale bars = 50 µm. ( C ) Quantification of PF density and branching density from images treated with angiotool. ( D ) Quantification of the percentage of YFP+ cells included in the VCS. N = 3 hearts per group. Mean ± SD; Student t -tests: * p < 0.05; ** p < 0.01; *** p < 0.001 Nkx2-5 ∆ VCS vs. control.

Article Snippet: Antibodies used in this study are specific to Nkx2-5 (Sc8697 Santa-Cruz, Dallas, TX, USA), GFP (AbD Serotec, Purchheim, Germany), RFP (Rockland, Pottstown, PA, USA), Contactin-2 (AF1714 R&D system, Minneapolis, MN, USA), Pecam-1 (MEC13.3-BD Pharmingen, San Jose, CA, USA), HCN4 (Millipore, Burlington, MA, USA), ETV-1 (Abcam, Cambridge, U.K.), Troponin I (Abcam) and WGA-Cy3 (Sigma, St. Louis, MO, USA).

Techniques: Immunofluorescence, Staining

Journal: Journal of Cardiovascular Development and Disease

Article Title: Nkx2-5 Loss of Function in the His-Purkinje System Hampers Its Maturation and Leads to Mechanical Dysfunction

doi: 10.3390/jcdd10050194

Figure Lengend Snippet: Ventricular conduction defects in Nkx2-5 ∆ VCS mutant hearts. ( A ) Immunofluorescence with Nkx2-5 and HCN4 or ETV1 and Contactin-2 or Cx40 , CNTN2 and WGA or Troponin I (TrI) and Cx43 on serial sagittal sections at the level the left Purkinje fibers from control (CTL) and Nkx2-5 ∆ VCS hearts. Scale bar = 100 µm. High magnifications of the selected area (rectangle) are presented below. Scale bar = 20 µm. ( B ) High magnifications at the level of LPF stained with WGA-cy3 and Nkx2-5 from 3- and 10-month-old control (CTL) and Nkx2-5 ∆ VCS hearts. Scale bar = 50 µm. ( C ) Nkx2-5 deletion was quantified by counting the percentage of Nk2-5 -positive cardiomyocytes per frame at the subendocardial surface of transverse sections from 3- and 10-month-old control (CTL) and Nkx2-5 ∆ VCS (∆VCS) mice. ( D ) Quantification of cardiomyocyte hypertrophy by counting the number of cardiomyocytes per frame on high-magnification images of transverse sections from 3- and 10-month-old control (CTL) and Nkx2-5 ∆ VCS (∆VCS) mice. n = 20–30 frames per heart; N = 3 mice per group; mean ± SD; two-way analysis of variance (ANOVA) followed by Sidak post hoc tests: ** p < 0.01; **** p < 0.0001 ΔVCS vs. control; $ p < 0.05; $$ p < 0.01, 3-month-old vs. 10-month-old hearts.

Article Snippet: Antibodies used in this study are specific to Nkx2-5 (Sc8697 Santa-Cruz, Dallas, TX, USA), GFP (AbD Serotec, Purchheim, Germany), RFP (Rockland, Pottstown, PA, USA), Contactin-2 (AF1714 R&D system, Minneapolis, MN, USA), Pecam-1 (MEC13.3-BD Pharmingen, San Jose, CA, USA), HCN4 (Millipore, Burlington, MA, USA), ETV-1 (Abcam, Cambridge, U.K.), Troponin I (Abcam) and WGA-Cy3 (Sigma, St. Louis, MO, USA).

Techniques: Mutagenesis, Immunofluorescence, Staining

Journal: Autophagy

Article Title: The autophagy-inducing kinases, ULK1 and ULK2, regulate axon guidance in the developing mouse forebrain via a noncanonical pathway

doi: 10.1080/15548627.2017.1386820

Figure Lengend Snippet: Abnormal axonal fasciculation in the Ulk1/2-deficient animals is associated with mislocalization of CNTN2 in the projection neurons. (A) Neuronal cell adhesion molecule distribution was unaltered in the ulk1/2-DKO brains compared to that in the controls. (B) The intensity of CNTN2 immunostaining (red) was dramatically decreased in distal CTAs of the ulk1/2-DKO brain at E18.5. All of the sections were counterstained with CHL1 cell adhesion molecule (green). (C) Western blot analyses of the extracts prepared from the cortex (proximal CTAs) and striatum (distal CTAs) confirmed normal expression of neuronal cell adhesion molecule but significantly decreased CNTN2 levels in the striatum. (D and E) Quantification of the NCAM1 and CNTN2 levels in control and ulk1/2-DKO cortex and striatum. Abbreviations: Cnt, controls; NCAM1, neuronal cell adhesion molecule 1; ns, not significant.*P < 0.05. Scale bars: 200 µm.

Article Snippet: After incubation with a 5% skim-milk block, blots were probed with antibodies directed against the following targets: ULK1 (Sigma Aldrich, A7481), SQSTM1 (Sigma Aldrich, P0067), LC3B (Novus Biologicals, NB100–2220), ATG13 (Sigma Aldrich, SAB4200100), RB1CC1 (Cell Signaling Technology, 12436), ATG14 (MBL, PD026), CNTN2 (R&D, AF4439), NCAM1 (Developmental Studies Hybridoma Bank, 5B8), and GAPDH (Sigma Aldrich, G9545).

Techniques: Immunostaining, Western Blot, Expressing

Journal: Molecular Neurodegeneration

Article Title: BACE1 activity regulates cell surface contactin-2 levels

doi: 10.1186/1750-1326-9-4

Figure Lengend Snippet: BACE1 cleaves contactin-2 close to its GPI domain. A) Schematic representation of partial amino acid sequence of contactin-2 indicating putative BACE1 cleavage site. B) Graphic representation of GPI-anchored and soluble contactin-2 proteins that were used in this study. C) Lentiviral-mediated overexpression of BACE1 increased contactin-2 and sAPP shedding in CHO cells stably expressing GPI-anchored human contactin-2 (CNTN2). (D) Release of soluble contactin-2 (sCNTN2) was not changed by BACE1 overexpression. E and F) Quantitative analysis of the secreted contactin-2 levels in C) and D) , respectively (Student t test; *, p < 0.05; n = 3 for each condition).

Article Snippet: Anti-human contactin-2 (MAB-1714) and anti-mouse contactin-2 (AF4439) antibodies were purchased from R&D Systems (Minneapolis, USA).

Techniques: Sequencing, Over Expression, Stable Transfection, Expressing

Journal: Molecular Neurodegeneration

Article Title: BACE1 activity regulates cell surface contactin-2 levels

doi: 10.1186/1750-1326-9-4

Figure Lengend Snippet: BACE1 inhibition reduces contactin-2 shedding. A) Western blot showing a sharp decrease in contactin-2 levels in CHO(CNTN2) cells treated with BACE1 Inhibitor IV. B) Quantitative analysis of the secreted contactin-2 levels in A) (Student t test; *, p < 0.05; n = 3 for each condition). C) Confocal microscopy images of cell surface contactin-2 levels in CHO cells expressing CNTN2. BACE1 Inhibitor IV induced a strong increase in contactin-2 surface levels (green). Nuclear staining with DAPI is shown in blue. Images are taken at identical settings.

Article Snippet: Anti-human contactin-2 (MAB-1714) and anti-mouse contactin-2 (AF4439) antibodies were purchased from R&D Systems (Minneapolis, USA).

Techniques: Inhibition, Western Blot, Confocal Microscopy, Expressing, Staining

Journal: Molecular Neurodegeneration

Article Title: BACE1 activity regulates cell surface contactin-2 levels

doi: 10.1186/1750-1326-9-4

Figure Lengend Snippet: BACE1 inhibition also decreases release of endogenous neuronal contactin-2. A) Western blot analysis of mouse primary neuronal cultures treated with 1 μM BACE 1 Inhibitor IV showed a significant decrease in contactin-2 and sAPP levels in the conditioned media. B) Quantitative analysis of secreted contactin-2 levels in A) (Student t test; **, p < 0.01; n = 3 for each condition).

Article Snippet: Anti-human contactin-2 (MAB-1714) and anti-mouse contactin-2 (AF4439) antibodies were purchased from R&D Systems (Minneapolis, USA).

Techniques: Inhibition, Western Blot

Journal: Molecular Neurodegeneration

Article Title: BACE1 activity regulates cell surface contactin-2 levels

doi: 10.1186/1750-1326-9-4

Figure Lengend Snippet: Site-directed mutagenesis of the putative BACE1 cleavage site specifically blocks contactin-2 shedding. A) Partial amino acid sequence of human contactin-2 indicating the putative BACE1 cleavage site. CNTN2-MM1008AA mutations were introduced to block BACE1 cleavage. B) Western blot analysis showing a dramatic reduction in contactin-2 release in the conditioned media of the cells expressing the BACE1-cleavage site mutant contactin-2 (CNTN2-MM1008AA). C) Quantitative analysis of secreted contactin-2 levels in B) .

Article Snippet: Anti-human contactin-2 (MAB-1714) and anti-mouse contactin-2 (AF4439) antibodies were purchased from R&D Systems (Minneapolis, USA).

Techniques: Mutagenesis, Sequencing, Blocking Assay, Western Blot, Expressing

Journal: Molecular Neurodegeneration

Article Title: BACE1 activity regulates cell surface contactin-2 levels

doi: 10.1186/1750-1326-9-4

Figure Lengend Snippet: BACE1 regulates surface levels of contactin-2 in mouse primary neuronal cultures. A) Western blot analysis showed that surface levels of contactin-2 were largely decreased by BACE1 overexpression in mouse primary neuronal cultures. B) Quantitative analysis of surface contactin-2 and the control N-cadherin levels in A) (Student t test; ***, p < 0.005; n = 3 for each condition). C) Western blot analysis showed that surface levels of contactin-2 were largely increased by BACE1 Inhibitor IV treatments. D) Quantitative analysis of surface contactin-2 and the control NrCAM levels in C) . (Student t test; ***, p < 0.005; n = 3 for each condition).

Article Snippet: Anti-human contactin-2 (MAB-1714) and anti-mouse contactin-2 (AF4439) antibodies were purchased from R&D Systems (Minneapolis, USA).

Techniques: Western Blot, Over Expression

Journal: Molecular Neurodegeneration

Article Title: BACE1 activity regulates cell surface contactin-2 levels

doi: 10.1186/1750-1326-9-4

Figure Lengend Snippet: Contactin-2 levels are decreased in AD brain samples inversely correlating with BACE1 levels or β-amyloid plaques. A) Western blot analysis showed that contactin-2 levels were decreased in AD brain samples as compared to age-matched controls. B) Quantitative analysis of contactin-2 levels in A) (**, p < 0.01, Student’s t test; n = 9 for AD, 8 for non-AD). Contactin-2 levels were inversely correlated with BACE1 levels ( C , p = 0.032, Person’s correlation test; n = 17) or amyloid plaque densities ( D , p = 0.0064) in these samples.

Article Snippet: Anti-human contactin-2 (MAB-1714) and anti-mouse contactin-2 (AF4439) antibodies were purchased from R&D Systems (Minneapolis, USA).

Techniques: Western Blot

Journal: Molecular Neurodegeneration

Article Title: BACE1 activity regulates cell surface contactin-2 levels

doi: 10.1186/1750-1326-9-4

Figure Lengend Snippet: BACE1 cleaves contactin-2 close to its GPI domain. A) Schematic representation of partial amino acid sequence of contactin-2 indicating putative BACE1 cleavage site. B) Graphic representation of GPI-anchored and soluble contactin-2 proteins that were used in this study. C) Lentiviral-mediated overexpression of BACE1 increased contactin-2 and sAPP shedding in CHO cells stably expressing GPI-anchored human contactin-2 (CNTN2). (D) Release of soluble contactin-2 (sCNTN2) was not changed by BACE1 overexpression. E and F) Quantitative analysis of the secreted contactin-2 levels in C) and D) , respectively (Student t test; *, p < 0.05; n = 3 for each condition).

Article Snippet: Anti-human contactin-2 (MAB-1714) and anti-mouse contactin-2 (AF4439) antibodies were purchased from R&D Systems (Minneapolis, USA).

Techniques: Sequencing, Over Expression, Stable Transfection, Expressing

Journal: Molecular Neurodegeneration

Article Title: BACE1 activity regulates cell surface contactin-2 levels

doi: 10.1186/1750-1326-9-4

Figure Lengend Snippet: BACE1 inhibition reduces contactin-2 shedding. A) Western blot showing a sharp decrease in contactin-2 levels in CHO(CNTN2) cells treated with BACE1 Inhibitor IV. B) Quantitative analysis of the secreted contactin-2 levels in A) (Student t test; *, p < 0.05; n = 3 for each condition). C) Confocal microscopy images of cell surface contactin-2 levels in CHO cells expressing CNTN2. BACE1 Inhibitor IV induced a strong increase in contactin-2 surface levels (green). Nuclear staining with DAPI is shown in blue. Images are taken at identical settings.

Article Snippet: Anti-human contactin-2 (MAB-1714) and anti-mouse contactin-2 (AF4439) antibodies were purchased from R&D Systems (Minneapolis, USA).

Techniques: Inhibition, Western Blot, Confocal Microscopy, Expressing, Staining

Journal: Molecular Neurodegeneration

Article Title: BACE1 activity regulates cell surface contactin-2 levels

doi: 10.1186/1750-1326-9-4

Figure Lengend Snippet: BACE1 inhibition also decreases release of endogenous neuronal contactin-2. A) Western blot analysis of mouse primary neuronal cultures treated with 1 μM BACE 1 Inhibitor IV showed a significant decrease in contactin-2 and sAPP levels in the conditioned media. B) Quantitative analysis of secreted contactin-2 levels in A) (Student t test; **, p < 0.01; n = 3 for each condition).

Article Snippet: Anti-human contactin-2 (MAB-1714) and anti-mouse contactin-2 (AF4439) antibodies were purchased from R&D Systems (Minneapolis, USA).

Techniques: Inhibition, Western Blot

Journal: Molecular Neurodegeneration

Article Title: BACE1 activity regulates cell surface contactin-2 levels

doi: 10.1186/1750-1326-9-4

Figure Lengend Snippet: Site-directed mutagenesis of the putative BACE1 cleavage site specifically blocks contactin-2 shedding. A) Partial amino acid sequence of human contactin-2 indicating the putative BACE1 cleavage site. CNTN2-MM1008AA mutations were introduced to block BACE1 cleavage. B) Western blot analysis showing a dramatic reduction in contactin-2 release in the conditioned media of the cells expressing the BACE1-cleavage site mutant contactin-2 (CNTN2-MM1008AA). C) Quantitative analysis of secreted contactin-2 levels in B) .

Article Snippet: Anti-human contactin-2 (MAB-1714) and anti-mouse contactin-2 (AF4439) antibodies were purchased from R&D Systems (Minneapolis, USA).

Techniques: Mutagenesis, Sequencing, Blocking Assay, Western Blot, Expressing

Journal: Molecular Neurodegeneration

Article Title: BACE1 activity regulates cell surface contactin-2 levels

doi: 10.1186/1750-1326-9-4

Figure Lengend Snippet: BACE1 regulates surface levels of contactin-2 in mouse primary neuronal cultures. A) Western blot analysis showed that surface levels of contactin-2 were largely decreased by BACE1 overexpression in mouse primary neuronal cultures. B) Quantitative analysis of surface contactin-2 and the control N-cadherin levels in A) (Student t test; ***, p < 0.005; n = 3 for each condition). C) Western blot analysis showed that surface levels of contactin-2 were largely increased by BACE1 Inhibitor IV treatments. D) Quantitative analysis of surface contactin-2 and the control NrCAM levels in C) . (Student t test; ***, p < 0.005; n = 3 for each condition).

Article Snippet: Anti-human contactin-2 (MAB-1714) and anti-mouse contactin-2 (AF4439) antibodies were purchased from R&D Systems (Minneapolis, USA).

Techniques: Western Blot, Over Expression

Journal: Molecular Neurodegeneration

Article Title: BACE1 activity regulates cell surface contactin-2 levels

doi: 10.1186/1750-1326-9-4

Figure Lengend Snippet: Contactin-2 levels are decreased in AD brain samples inversely correlating with BACE1 levels or β-amyloid plaques. A) Western blot analysis showed that contactin-2 levels were decreased in AD brain samples as compared to age-matched controls. B) Quantitative analysis of contactin-2 levels in A) (**, p < 0.01, Student’s t test; n = 9 for AD, 8 for non-AD). Contactin-2 levels were inversely correlated with BACE1 levels ( C , p = 0.032, Person’s correlation test; n = 17) or amyloid plaque densities ( D , p = 0.0064) in these samples.

Article Snippet: Anti-human contactin-2 (MAB-1714) and anti-mouse contactin-2 (AF4439) antibodies were purchased from R&D Systems (Minneapolis, USA).

Techniques: Western Blot

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: eLife

Article Title: p75NTR prevents the onset of cerebellar granule cell migration via RhoA activation

doi: 10.7554/eLife.79934

Figure Lengend Snippet: ( A ) Mouse developmental expression of p75NTR (green), Pax6 (magenta), DCX (white), and Dapi (blue) at the indicated ages. Note the high level of p75NTR in the meninges as well as the developing granule cell progenitors. Yellow arrows indicate the M – meninges, RL – rhombic lip, EGL – external granule layer. ( B ) High magnification of the insets showed in A. Cells expressing p75NTR (arrows), migrating cells negative for p75NTR (arrowheads). ( C ) Expression of p75NTR (green), Ki67 (magenta), and Dapi (blue) in the cerebellum of P7 mouse pups. ( D ) Immunohistochemistry of the expression of p75NTR (green), TAG1 (magenta), and Dapi (blue) in the cerebellum of P7 mouse pups. ( E ) Immunohistochemistry of the expression of p75NTR (green), DCX (magenta), and Dapi (blue) in the cerebellum of P7 mouse pups. ( F ) High magnification of the inset showed in E. The tissue shown in all the figures were obtained from mice.

Article Snippet: Antibodies used were: Ki67 (Abcam 15580, RRID: AB_443209 , 1:500), anti-p75 (R&D AF367, RRID: AB_2152638 , 1:500), anti-p75 (Millipore MAB365, RRID: AB_2152788 , 1:1000), anti-PAX-6 (BD Bioscience, RRID: AB_397991 , 1/500), anti-DCX (Abcam 18723, RRID: AB_732011 , 1/500), and anti-TAG1 (R&D Systems AF1714, RRID: AB_2245173 , 1/500).

Techniques: Expressing, Immunohistochemistry