goats  (R&D Systems)

Journal: Nature Communications

Article Title: Transcriptional regulation of the postnatal cardiac conduction system heterogeneity

doi: 10.1038/s41467-024-50849-1

Figure Lengend Snippet: a Representative epifluorescence image of a P4 CCS-reporter mouse ( Cntn2 Cre/+ ; Rosa26 tdTomato/+ ) heart showing tdTomato-marked sinoatrial node, His bundle, bundle branches and Purkinje fibers. Scale bar = 1 mm. Imaging was performed on more than five hearts . b (top) Schematic overview of scRNA-seq experiment for the CCS profiling. Neonatal mouse cardiomyocytes were isolated from postnatal days 1, 2, and 4 (P1, P2, and P4) CCS-reporter mouse hearts, and tdTomato-positive CCS cells were purified using fluorescence-activated cell sorting (FACS). P1, P2, and P4 cells were barcoded separately using MULTI-seq, followed by 10x Genomics scRNA-seq. (bottom) Schematic overview of spatial transcriptomics of the CCS using GeoMx Digital Spatial Profiler (DSP). Cryosections of P4 CCS-reporter mouse hearts were collected on a slide, followed by the selection of regions of interest using segmentation and sequencing. Results from scRNA-seq and ST were integrated for analysis. Created with BioRender.com. c Uniform Manifold Approximation and Projection (UMAP) of scRNA-seq showing 8 CCS and 1 non-CCS clusters. A pie chart (top, left) shows the number of cells in each cluster. d Principal component analysis (PCA) of the CCS and non-CCS regions profiled by spatial transcriptomics (ST). e Expression profiles of the established CCS and non-CCS marker genes in each cluster of scRNA-seq data. f Expression profiles of the established CCS and non-CCS marker genes in each collected region of ST data. g Correlation analysis of CCS components between scRNA-seq and ST data. SAN sinoatrial node, cAVN compact atrioventricular node, LNB lower nodal bundle, HIS His bundle, PBB proximal bundle branch, PLBB proximal left bundle branch, PRBB proximal right bundle branch, DBB distal bundle branch, DLBB distal left bundle branch, DRBB distal right bundle branch, PF Purkinje fibers, RPF right Purkinje fibers, LPF left Purkinje fibers, RAA right atrial appendage, LENDO left ventricular endo-myocardium, LEPI left ventricular epi-myocardium, IVS-B interventricular septum (Base), IVS-A interventricular septum (Apex), RV right ventricle.

Article Snippet: The sections were then incubated overnight at 4 °C with the following primary antibodies: chicken anti-RFP (Rockland Immunochemicals, 600-901-379, lot #42649, 1:100), goat anti-Cntn2 (R&D Systems, AF4439, lot #CDSO0219101, 1:50), rabbit anti-Gnao1 (Thermo Fisher Scientific, PA5-30044, lot #YH4027897A, 1:100), rabbit anti-Scn10a (Alomone Labs, ASC-016, lot #, ASC016AN2550, 1:50), rabbit anti-Igfbp7 (Abcam, ab74169, lot #GR242021-1, 1:50), rabbit anti-S100a6 (Abcam, ab181975, lot #GR3438351-1, 1:100), rabbit anti-Nppa (EMD Millipore, ab5490, lot #Q3015715, 1:250), mouse anti-Shox2 IgG2a (Santa Cruz Biotechnology, sc-81955, lot #B2822, 1:50) or rabbit anti-Vsnl1 (GeneTex, GTX115039, lot #40247, 1:100), rabbit anti-Ppp1r17 (Thermo Fisher Scientific, PA5-61599, lot #YH4028316B, 1:200).

Techniques: Imaging, Isolation, Purification, Fluorescence, FACS, Selection, Sequencing, Expressing, Marker

Journal: Nature Communications

Article Title: Transcriptional regulation of the postnatal cardiac conduction system heterogeneity

doi: 10.1038/s41467-024-50849-1

Figure Lengend Snippet: Immunofluorescence (IF) and fluorescence in situ hybridization (FISH) staining were performed in P4 Cntn2 Cre/+ ; Rosa26 tdTomato/+ heart sections. a Gnao1 IF staining in tdTomato-positive SAN and cAVN, but not LNB; b Prdm6 FISH staining in the cAVN, but not LNB; c Ppp1r17 IF staining in the LNB and HIS; d S100a6 IF staining in the HIS; e Igfbp7 IF staining in the HIS and proximal LBB; f Nppa IF staining in the DBB; g Mpped2 FISH staining in the VCS. scale bars = 50 μm. The figures shown are representative of at least three independently repeated IF experiments or two FISH experiments. SAN sinoatrial node, cAVN compact atrioventricular node, LNB lower nodal bundle, HIS His bundle, LBB left bundle branch, RBB right bundle branch, LPF left Purkinje fibers, RPF right Purkinje fibers.

Article Snippet: The sections were then incubated overnight at 4 °C with the following primary antibodies: chicken anti-RFP (Rockland Immunochemicals, 600-901-379, lot #42649, 1:100), goat anti-Cntn2 (R&D Systems, AF4439, lot #CDSO0219101, 1:50), rabbit anti-Gnao1 (Thermo Fisher Scientific, PA5-30044, lot #YH4027897A, 1:100), rabbit anti-Scn10a (Alomone Labs, ASC-016, lot #, ASC016AN2550, 1:50), rabbit anti-Igfbp7 (Abcam, ab74169, lot #GR242021-1, 1:50), rabbit anti-S100a6 (Abcam, ab181975, lot #GR3438351-1, 1:100), rabbit anti-Nppa (EMD Millipore, ab5490, lot #Q3015715, 1:250), mouse anti-Shox2 IgG2a (Santa Cruz Biotechnology, sc-81955, lot #B2822, 1:50) or rabbit anti-Vsnl1 (GeneTex, GTX115039, lot #40247, 1:100), rabbit anti-Ppp1r17 (Thermo Fisher Scientific, PA5-61599, lot #YH4028316B, 1:200).

Techniques: Immunofluorescence, Fluorescence, In Situ Hybridization, Staining

Journal: Science Advances

Article Title: A global gene regulatory program and its region-specific regulator partition neurons into commissural and ipsilateral projection types

doi: 10.1126/sciadv.adk2149

Figure Lengend Snippet: ( A ) The schematic shows the PN formation in an E16.5 hindbrain. Red dashed lines mark the rostral (r) and caudal (c) span of the sections shown on the right. PN is visualized by Barhl1 IHC. PN neurons form a nucleus adjacent to the ventral midline (arrows) ( n = 3) in the control but were laterally positioned (arrowheads) indicating failure to approach the ventral midline in the mutants ( n = 3). ( B to D ) Coronal sections from the spinal cord (B), hindbrain (C), and midbrain (D) at E11.5 were subjected to Tag1 and NF double IHC with the Tag1 labeling the commissural axons and the NF signals depicting the general axonal patterns. The DAPI counterstain indicates 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. The double mutant showed a complete lack of ventral commissures (hollow arrows), in comparison to the control (filled arrows). Scale bars, 200 μm [(A) to (D)].

Article Snippet: The primary antibodies used were rabbit anti-Barhl1 polyclonal antibody (Atlas Antibodies; HPA004809, Sigma-Aldrich; 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–NF-160kD monoclonal antibody (clone RMO-270; Thermo Fisher Scientific, 13-0700; 1:500), rat anti-L1CAM monoclonal antibody (clone 324; Merck Millipore, 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; Merck Millipore, MAB1585; 1;200), rabbit anti-Lim1/Lhx1 antibody (Abcam, ab229474; 1:250), rabbit anti-Lhx2 (Invitrogen, PA5-78287; 1:250), rat anti-HA (Roche; clone3F10; 1:100), guinea pig anti-Isl1 antibody (a gift from Y. Tanabe, Kyoto University, Japan), and chick anti-GFP polyclonal antibody (Abcam, ab13970; 1:1500).

Techniques: Control, Labeling, Mutagenesis, Comparison