Tcs sp2 confocal microscope system

Small round skin fragments (6 mm in diameter) were harvested from the back of mice with a disposable punch biopsy (Kai Industries, Gifu, Japan). Skin samples were incubated in Dulbecco’s modified Eagle medium containing 4% hyaluronate and 5% fetal bovine serum for 6 hours under 1% (hypoxia), 6% (normoxia), or 20% (hyperoxia) oxygen (n = 3 for each group). After washing with phosphate-buffered saline, they were incubated with Hoechst33342 (Dojindo, Kumamoto, Japan) and propidium iodide (PI; Sigma-Aldrich, St. Louis, Mo.) at 37°C for 30 minutes. The stained samples were evaluated under a TCS SP2 confocal microscope system (Leica, Heerbrugg, Switzerland). Ten serial images of the dermis were obtained at 3-μm intervals and were processed to produce a surface-rendered, 30-μm-thick, three-dimensional image. Numbers of all nuclei (Hoechst-positive) and dead nuclei (Hoechst- and PI-positive) were counted.

This method was similar to previous work from our laboratory (Fan et al., 2011 (link)). Briefly, the slides containing the sections of LC regions were pre-incubated in 5% bovine serum albumin in phosphate-buffer saline (PBS) supplemented with 0.2% Triton-X 100. These slides were further exposed to primary monoclonal antibody solution (for DBH: 1:2000, ab19353, RRID: AB_73185, Abcam, Cambridge, MA, USA; for TH: 1:1000, MA1-24654, RRID: AB_79566, ThermoFisher Scientific, Waltham, MA, USA) overnight at 4°C. After washing, slides were then probed with 2nd antibodies (for DBH: Alexa Fluor 488-conjugated goat anti-rabbit IgG; for TH: Alexa Fluor 488-conjugated Goat anti-mouse IgG; both from Abcam, Cambridge, MA, USA), followed by 4 time rinses with 0.1 M PBS. A Leica TCS SP2 confocal microscope system (Leica Microsystems Inc., Bannockburn, IL, USA) was used to observe and acquire immunofluorescence labeling. These images were quantitatively quantified using ImageJ software (Rasband, US National Institutes of Health, Bethesda, http://rsbweb.nih.gov/ij, 2010). Non-immunoreactive portions of brain sections adjacent to the LC region were taken as the reference background levels.