Light microscope eclpse 80i

On days 7 and 14, the rats were sacrificed to harvest the wound tissues. The tissues were fixed in cold 4% paraformaldehyde in 0.01 M phosphate-buffered saline (pH 7.4) overnight. Afterward, the tissues were dehydrated and embedded into paraffin. Then, the tissues were cut into 5 μm thickness slices with a microtome (LEICA RM2235, Germany). The tissue slides were finally processed for the hematoxylin and eosin staining (H&E) and Masson’s trichrome staining (MTS). The staining images were obtained with a Nikon light microscope (ECLPSE 80i, Nikon, Japan) for wound healing evaluation. For the quantification of collagen density, the MTS images were split into different colors (red, blue, and green) with the color deconvolution plugin in the Image-Pro Plus 6.0 software. The blue positive area and total staining area were measured by the software, and the collagen density was calculated according to the following equation: Collagen density = (the blue-stained area/the total area) × 100%. Three staining images for each group were selected for the quantification and carry out the statistical analysis.

After deparaffinization and rehydration, skin tissue sections were submerged in 3% hydrogen peroxide at room temperature for 15 min to inactivate the endogenous peroxidase of the sections, and then soaked in 5% BSA to block nonspecific binding sites at 37 °C for 30 min. Subsequently, sections were incubated at 4 °C overnight with antibody against CD31 (1:100), and TNF-α (1:300). The sections were then rinsed with PBS, and incubated at 37 °C for 60 min with biotinylated secondary antibodies that diluted with PBS (1:1000), corresponding to the first antibodies, respectively. Finally, the reaction was stopped by a DAB Chromogen Kit for all sections for 8 s to 3 min, and the tissue sections were counterstained with hematoxylin and mounted with neutral resin. Images were captured by a Nikon light microscope (ECLPSE 80i, Nikon, Japan), and analyzed by an Image-Pro Plus software (Nikon, Tokyo, Japan).