Mouse anti col 1 antibody

Immunohistochemistry was performed on 5-µm-thick paraffin-embedded tissue sections. The sections were dewaxed, and endogenous peroxidase activity was quenched with 3% hydrogen peroxide for 10 min, followed by blocking with the corresponding serum from a secondary antibody raised animal species for 1 hour. Slides were then incubated overnight at 4°C with the primary antibody mouse anti-Col-I antibody (1∶1000, abcam, USA) diluted in the blocking serum. After several washes with PBS, the tissue sections were incubated with peroxidase-conjugated goat anti-mouse polyclonal antibody (DAKO, Carpinteria, CA) at a dilution of 1∶200 in PBS for 30 min at 37°C. After several washes in PBS, the signals on the tissue were revealed by incubating with DAB in PBS for 5–10 min, followed by hematoxylin counterstaining. All the tissue sections were observed under a microscope (Nikon, Japan) at a 400× magnification, and 5 randomly chosen fields of dermis were photographed for each wound. The sizes of areas of collagen deposition were estimated using the Image Pro Plus 6.0 software. The relative density of collagen in each group was calculated by normalizing to the unwounded skin collagen density.

Total cellular protein was extracted with RIPA lysis buffer, and protein concentration of lysates was measured by BCA Protein Assay Kit (Thermo, Waltham, USA). The protein samples were stored at −80°C for further analysis. Subsequently, equal amounts of proteins were separated by 10% SDS-PAGE gel and then transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, USA). After blocking in 5% non-fat milk in TBST buffer for 1 h at room temperature, the membranes were incubated with three primary antibodies, respectively, including mouse anti-vinculin antibody (Sigma; V9131; 1:1500), mouse anti-Col-I antibody (Abcam; ab88147; 1:2000), mouse anti-α-SMA antibody (Sigma; A5228; 1:2000), or mouse anti-β-actin antibody (Affinity; T0022; 1:3000) at 4°C overnight, followed by incubation with HRP-linked goat anti-mouse IgG (H + L) (Affinity; S0002; 1:3000) at 37°C for 1 h. After washing the membranes with TBST, the band signals were visualized by using Affinity® ECL Reagent. Finally, the membranes were exposed and analyzed by using Luminescent Image Analyzer (Amersham, Uppsala, Sweden). The results of each blot were normalized against β-actin protein expression.