COMP-angiopoietin 1 Gene Transfer Enhances Cutaneous Wound Healing: MATERIALS AND METHODS part 2
Histology and immunohistochemistry
Three wounds on the back of each rat were sequentially removed from the site at days 3, 7 and 14 after surgery. Each specimen was divided into two equal parts. One half of the specimens were fixed in 4% paraformaldehyde, washed in tap water, dehydrated in a series of graded ethanol solutions, cleared in xylene, and then embedded in paraffin for light microscopic examination. The sections (4 pm thickness) were then mounted on glass slides, hydrated with distilled water, and subjected to hematoxylin-eosin staining (Vector Laboratories, Burlingame, CA, USA).
The other half of each specimen was embedded in OCT compound (Sakura, Tokyo, Japan) for immunostaining. The skin specimens were prepared for immunohistochemical staining as previously described. Briefly, five-micrometer cryostat sections were cut on a cryostat microtome (Leica Microsystems AG, Wetzlar, Germany), and then fixed in acetone for 15 min. After three washes in phosphate buffered saline (PBS), the sections were incubated for 10 minutes in methanol with 0.3% hydrogen peroxide to block the endogenous peroxi- dase activity. Nonspecific antibody binding was blocked by incubating the sections with 10% normal donkey serum (Jackson Immuno Research Laboratories, West Grove, PA, USA) for 60 min. The sections were incubated with mouse anti-rat PECAM-1/CD31 monoclonal antibodies (1 : 100 in PBS, Cemicon, CA, USA) overnight at 4oC. Subsequently, the tissue sections were incubated with secondary antibodies (1 : 300 in PBS, Jackson Immuno Research Laboratories) for 60 min. As controls, the same skin specimens were incubated with an isotype-matched antibody. As a negative control, skin samples were incubated with PBS without the primary antibodies. The color reaction of the treated tissue was carried out using the substrate 3-amino-9-ethylcarbazole (Vector Laboratories) for 10 min. The skin sections were also counterstained with hematoxylin (Vector Laboratories) for 1 to 2 min.
official canadian pharmacy
The number of blood vessels with definite lumens or with red blood cells in it was counted under a light microscope under the high power view (x200) by two independent dermatologists who were without knowledge of the previous treatment, and then the mean number and standard deviation of vessels in the Ade-COMP-Ang 1-treated and Ade- LacZ-treated groups were calculated.
Statistical analysis
The data were analyzed by two-way repeated measures analysis of variance (ANOVA) tests, two samples t-tests and paired t-tests were performed for statistical analysis. The data were analyzed by the statistical program SPSS for Windows 10.0 (SPSS Inc., Chicago, IL, USA). Statistical significance was defined as p<0.05.
