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