Mice (6 weeks old) containing one copy of the CTGF/E-GFP allele were anesthetized by intraperitoneal injection of 90 μg ketamine plus 10 μg xylazine/g, and their back skin was shaved, depilated with commercially available hair removal cream (Nair; Church and Dwight, Princeton, NJ, USA) and cleaned with alcohol. Using a sterile 4-mm biopsy punch, four bilateral full-thickness skin wounds were created on the dorsorostral back skin. Wounds were separated by a minimum of 6 mm of uninjured skin. Mice were killed by CO2 euthanasia after 0, 3 and 7 days post-wounding and wound tissue biopsies were collected for immunoflourescence.
Wound tissue sections (0.5 μm) were cut using a microtome (Leica, Richmond Hill, ON, Canada) and collected on Superfrost Plus slides (Fisher Scientific, Ottawa, ON, Canada). Sections were then de-waxed in xylene and rehydrated by successive immersion in descending concentrations of alcohol. Sections were then subjected to single or double immunofluorescence. Briefly, tissue sections were incubated with mouse serum for 30 min and washed with phosphate-buffered saline (PBS). Sections were then incubated with primary antibodies for 1 h at room temperature under humidified conditions. Primary antibodies used alone (single immunofluorescence) or in combination (double immunofluorescence) were: rabbit anti-green fluorescent protein (anti-GFP; 1:100 dilution, Invitrogen, Burlington, ON, Canada), mouse anti-NG2 (pericyte marker, 1:100 dilution, Chemicon, Millipore, Billerica, MA, USA), mouse anti-alpha-smooth muscle actin (α-SMA, 1:100 dilution, Sigma, St Louis, MO, USA). Double immunofluorescence for α-SMA and NG2 was performed using rabbit polyclonal antibody for α-SMA (Abcam, Cambrodge, MA, USA) and mouse antibody for NG2. Both rabbit and mouse antibodies for α-SMA showed identical staining of cells and tissues in our system. After primary antibody incubation, sections were then washed with PBS and incubated with appropriate fluorescent secondary antibodies (Jackson Immunoresearch, West Grove, PA, USA) for 1 h at room temperature. Sections were then washed with PBS and mounted using 4',6-diamidino-2-phenylindole (DAPI) and photographed using a Zeiss fluorescence microscope and Northern Eclipse software (Empix, Missassagua, ON, Canada). Six independent fields were examined per data point.
The tissue expression of GFP/CTGF and α-SMA on days 0, 3 and 7 post-wounding was graded on a scale of 0–3 by three blinded observers; 0 signifies no staining, 1 signifies very little staining, 2 signifies moderate staining, 3 signifies extensive staining.
Sections undergoing double immunofluorescence were photographed at 10 × (for whole tissue section) and 60 × (for cells) magnifications. To detect number of α-SMA, GFP/CTGF or NG2 positive cells in and around wound area, two planes were chosen: (a) cells in the centre of the wound, and (b) infiltrating cells at wound edges. At each plane, the total number of cells/mm2 were counted. Subsequently, the number of α-SMA, GFP/CTGF and NG2 positive cells/mm2 were counted and expressed as percentage positive cells at each plane.