Journal: Journal of periodontal research
Article Title: Type 1 diabetes predisposes to enhanced gingival leukocyte margination and macromolecule extravasation in vivo
Figure Lengend Snippet: In vivo assessment of gingival vascular permeability and leukocyte margination. Rhodamine 6G (R6G; leukocyte labeling) and FITC– dextran (vasculature visualization and tracer molecule) or FITC–LY6G (PMN labeling) were injected intravenously, and intravital microscopy was performed at a controlled body temperature as specified in the Material and methods section. The black arrows indicate the outer side of the vessel wall. (A) The FITC–dextran is retained in the gingival vasculature of a WT mouse. (B) Increased FITC–dextran extravasation in Akita gingiva within the same time period. (C) Rhodamine 6G-positive leukocytes inside gingival postcapillary venules. The FITC–dextran (green) and R6G (red) images were merged offline to obtain this picture. (D) Overlapping images of R6G-positive (red) and LY6G-positive (green) cells indicate that the majority of leukocytes 2 h after tumor necrosis factor α (TNFα) stimulation are polymorphonuclear leukocytes (PMNs; yellow). The white arrows indicate intravascular leukocytes as follows: 1,rolling leukocyte (red); 2, rolling PMN (green); and 3, attached PMN (yellow). Scale bar represents 50 μm; original magnification ×400.
Article Snippet: Rhodamine 6G (R6G; Molecular Probes; Invitrogen, Carlsbad, CA, USA), fluorescein isothiocyanate–dextran (FITC–dextran; molecular weight, 150 kDa), Wright-Giemsa, zymosan A solution (Sigma-Aldrich, St. Louis, MO, USA), FITC-anti-LY6G, anti-PSGL1, anti-CD11a antibodies (BD Biosciences, San Jose, CA, USA), anti-P-selectin, anti-L-selectin antibodies (Santa Cruz, CA, USA) ketamine, xylazine, isoflurane, tumor necrosis factor α (TNFα; Roche Diagnostics), sterile saline solution (0.9% NaCl), phosphate-buffered saline (PBS; Invitrogen Life Technologies, Carlsbad, CA, USA), Bradford protein assay (Bio-Rad, Hercules, CA, USA), 5–0 silk surgical suture (Benco Dental, Pittston, PA, USA), FACScan flow cytometer and Cell Quest computer Software (Becton-Dickinson, Franklin Lakes, NJ, USA), FITC and tetramethylrhodamine isothiocyanate (TRITC) filter sets (Chroma Technology, Rockingham, VT, USA), luminal-enhanced chemiluminescence detection reagent (Cell Signaling Technology, Danvers, MA, USA), Zeiss Axiovert 200 inverted epifluorescent microscope equipped with a Sony DFW-X700 digital video camera, Sonycap (Sony Corporation, Tokyo, Japan) and Olympus MicroSuite software were obtained commercially, as indicated.
Techniques: In Vivo, Permeability, Labeling, Injection, Intravital Microscopy