Journal: bioRxiv

Article Title: Tiered Sympathetic Control of Cardiac Function Revealed by Viral Tracing and Single Cell Transcriptome Profiling

doi: 10.1101/2023.01.18.524575

Figure Lengend Snippet: Validation of retrograde tracing of AAVs from myocardium (Heart injections). A , Cross-section of the heart labeled with AAV-GFP (green) and cardiac troponin I. Scale bar: 1000 μm . B , PGP9.5 staining in nerve fibers of a heart section. C , AAV-GFP labeling in nerve fibers of the heart section. D , Merge image shows the co-labeling of PGP9.5 and AAV-GFP in nerve fibers of the heart-section that travel to the stellate ganglia. n = 3 mice. Scale bar: 50 μm

Article Snippet: The primary antibodies were: sheep anti-TH (Millipore, #ab1542, 1:200), rabbit anti-CNTN5 (Thermo Fisher Scientific, #PA5-59486, 1:30), rabbit anti-NPY (Cell Signaling Technology, #11976S, 1:200), goat anti-NPY (Novus Biologicals, #NBP1-46535, 1:200), rabbit anti-VIP (ThermoFisher Scientific, #PA5-78224, 1:100), rabbit anti-KCNT1/SLACK[N3/26] (Abcam, #ab94578, 1:50), goat anti-NGFR (R&D System, #AF1157, 1:100), goat anti-vimentin (Millipore-Sigma, #V4630, 1:400), rabbit anti-calmodulin (Novus Biologicals, #NBP2-67413, 1:100), rabbit anti-SCTR (Thermo Fisher Scientific, #PA5-76698, 1:25), rat anti-SST (Millipore-Sigma, #MAB354, 1:50), goat anti-FABP7 (R&D system, #AF3166, 1:100), rabbit anti-CASZ1 (Thermo Fisher Scientific, #PA5-55800, 1:100), goat anti-cardiac troponin I (Abcam, #ab56357, 1:200), and rabbit anti-PGP9.5 (Abcam, #ab108986, 1:400) used for the specific key gene markers.

Techniques: Retrograde Tracing, Labeling, Staining

Journal: bioRxiv

Article Title: Tiered Sympathetic Control of Cardiac Function Revealed by Viral Tracing and Single Cell Transcriptome Profiling

doi: 10.1101/2023.01.18.524575

Figure Lengend Snippet: Transcriptomic and electrophysiological properties of Npy high vs. Npy low/neg neurons. A ; B ; C ; D , Distribution of different ion channels associated with Npy high and Npy low/neg expressing cells: Potassium channels ( A ); Sodium channels ( B ); Calcium channels ( C ); and several other chloride, acid sensing ion channels ( D ) in high, medium, and low Npy expressing cells. E , Confocal image of a fixed stellate ganglion whole mount. PGP9.5 (red), NPY-GFP+ (green), and Neurobiotin (yellow). Magnified images from boxed region appear in right hand panels. F , NPY+ cells identified by epifluorescence during targeting with intracellular microelectrodes. Left panels DIC image ( i , iv ), right panel GFP (green) ( ii , v ). iii , vi; Membrane potential tracings from a GFP+ ( iii ) and a GFP- ( vi ) cells in response to 500ms hyperpolarizing and depolarizing pulses. Resting membrane potential for both cells was more or less similar to ~ −52.45mV. Dash is 0 mV. G ; H ; I , Summary electrophysiology data for the NPY+ (n = 11 mice) and NPY- cells (n = 11 mice). GFP+ cells had similar current-voltage relationship ( G ) but showed greater membrane excitability in response to intracellular depolarization ( H ). Data are shown as means ±SEM; ***p<0.001 compared with NPY- cells. The raster plot ( I ) illustrates time of peak action potential amplitude in response to 500ms, 500pA depolarizing current. Note the greater number of APs and shorter interspike interval in NPY+ cells that reveal increased neuronal excitability. A two-way ANOVA followed by Sidak’s multiple comparison test was used for statistical analyses.

Article Snippet: The primary antibodies were: sheep anti-TH (Millipore, #ab1542, 1:200), rabbit anti-CNTN5 (Thermo Fisher Scientific, #PA5-59486, 1:30), rabbit anti-NPY (Cell Signaling Technology, #11976S, 1:200), goat anti-NPY (Novus Biologicals, #NBP1-46535, 1:200), rabbit anti-VIP (ThermoFisher Scientific, #PA5-78224, 1:100), rabbit anti-KCNT1/SLACK[N3/26] (Abcam, #ab94578, 1:50), goat anti-NGFR (R&D System, #AF1157, 1:100), goat anti-vimentin (Millipore-Sigma, #V4630, 1:400), rabbit anti-calmodulin (Novus Biologicals, #NBP2-67413, 1:100), rabbit anti-SCTR (Thermo Fisher Scientific, #PA5-76698, 1:25), rat anti-SST (Millipore-Sigma, #MAB354, 1:50), goat anti-FABP7 (R&D system, #AF3166, 1:100), rabbit anti-CASZ1 (Thermo Fisher Scientific, #PA5-55800, 1:100), goat anti-cardiac troponin I (Abcam, #ab56357, 1:200), and rabbit anti-PGP9.5 (Abcam, #ab108986, 1:400) used for the specific key gene markers.

Techniques: Expressing

Journal: The Journal of Comparative Neurology

Article Title: Using tissue clearing and light sheet fluorescence microscopy for the three‐dimensional analysis of sensory and sympathetic nerve endings that innervate bone and dental tissue of mice

doi: 10.1002/cne.25582

Figure Lengend Snippet: Details of the primary and secondary antibodies used in this study.

Article Snippet: Protein gene product 9.5 (PGP9.5) , 15 amino synthetic peptide located near the C‐terminus of human PGP9.5 (UCHL1) , Cedarlane Laboratories; rabbit polyclonal; Cat CL7756AP, RRID: AB_2792979 , Doran et al. ( ) , 1:500.

Techniques:

Journal: Scientific Reports

Article Title: 3D-Imaging of Whole Neuronal and Vascular Networks of the Human Dental Pulp via CLARITY and Light Sheet Microscopy

doi: 10.1038/s41598-019-47221-5

Figure Lengend Snippet: Schematic of the optimized CLARITY process to clear human dental pulp. Freshly extracted dental pulp samples were embedded in formaldehyde, acrylamide and bisacrylamide monomers. ( A ) Amine groups from proteins were covalently linked to monomers and polymerized into a hydrogel. ( B ) Lipids are passively removed with a SDS buffer. ( C ) The tissue-polymer hybrids are stained with CD31 and PGP9.5 antibodies ( D ), mounted in an agarose plug ( E ), imaged with a light sheet microscope ( F ) and images were rendered and quantified using Arivis and Imaris softwares ( G ) showing several possibilities of image visualization, as maximum intensity projection surface covering and neural bundles identification.

Article Snippet: 1% Triton X-100 (Sigma) in PBS) for 24 h. For endothelial stain samples were incubated with primary mouse monoclonal antibody anti-CD31 (clone 89C2, # 3528, Cell Signaling), 1:100 in PBST and for neural stain incubation was with primary rabbit polyclonal anti-PGP9.5 (Accurate Chemical and Scientific Corp., Westbury, NY) or UCHL1/PGP 9.5 (rabbit polyclonal, Proteintech cat # 14730 1-AP) for 48 h at 37 °C.

Techniques: Staining, Microscopy

Journal: Scientific Reports

Article Title: 3D-Imaging of Whole Neuronal and Vascular Networks of the Human Dental Pulp via CLARITY and Light Sheet Microscopy

doi: 10.1038/s41598-019-47221-5

Figure Lengend Snippet: Whole innervation of a human premolar from a mesio-distal view ( A ), a palatal-vestibular perspective ( B ), an occlusal ( C ) and an apical-coronal view ( D ). In the high-magnification images from the crown it is possible to observe several nerve bundles that run straightforward in the coronal direction ( E ) splitting (*) down to the level of a single nerve fiber from the plexus of Raschkow directing towards the dentin site. ( F ) In the furcation level it can be observed thick bundles that come from the root and start to split (*) determining the site-specific innervation of the pulp chamber ( G , H ) (Immunofluorescence, PGP9.5 - green, CD31 – red).

Article Snippet: 1% Triton X-100 (Sigma) in PBS) for 24 h. For endothelial stain samples were incubated with primary mouse monoclonal antibody anti-CD31 (clone 89C2, # 3528, Cell Signaling), 1:100 in PBST and for neural stain incubation was with primary rabbit polyclonal anti-PGP9.5 (Accurate Chemical and Scientific Corp., Westbury, NY) or UCHL1/PGP 9.5 (rabbit polyclonal, Proteintech cat # 14730 1-AP) for 48 h at 37 °C.

Techniques: Immunofluorescence

Journal: Scientific Reports

Article Title: 3D-Imaging of Whole Neuronal and Vascular Networks of the Human Dental Pulp via CLARITY and Light Sheet Microscopy

doi: 10.1038/s41598-019-47221-5

Figure Lengend Snippet: Neurovascular bundles in 3D at the furcation level showing the blood vessels ranging from 4 to 30 µm. ( A–C ) The focal planes 1 and 2 have a difference of 100 µm in the Z-distance, and illustrate the complexity of dental pulp neural and vascular interplay. Some blood vessels were embedded into the neural structures and others were either innervated by single nerve fibers ( D–F ) or did not show innervation at the level of the imaged site. ( F ) (Immunofluorescence, PGP9.5 - green, CD31 – red, confocal 20x objective).

Article Snippet: 1% Triton X-100 (Sigma) in PBS) for 24 h. For endothelial stain samples were incubated with primary mouse monoclonal antibody anti-CD31 (clone 89C2, # 3528, Cell Signaling), 1:100 in PBST and for neural stain incubation was with primary rabbit polyclonal anti-PGP9.5 (Accurate Chemical and Scientific Corp., Westbury, NY) or UCHL1/PGP 9.5 (rabbit polyclonal, Proteintech cat # 14730 1-AP) for 48 h at 37 °C.

Techniques: Immunofluorescence