Ab1827

Cells on slides were fixed in 4% paraformaldehyde/PBS for 30 minutes and rinsed with PBS. After permeabilization with 0.1% Triton X-100/PBS for 10 minutes, the slides were blocked in 3% BSA for 30 minutes. The slides were then incubated with the primary antibodies (anti-MMP1, Catalogue number ab52631, Abcam, 1:100 dilution; anti-TIMP1, ab1827, Abcam, 1:100 dilution; anti-TGFB1, BA0290, Boster, 1:50 dilution; anti-TGFB2, BA0292, Boster, 1:50 dilution; 100μl each) for 2 hours at room temperature. After washing with PBS three times, the slides were incubated with Fluorescein isocyanate (FITC)-conjugated anti-rabbit antibody at room temperature for 1 hour. After mounting (UltraCruz Mounting Medium, SC-24941; Santa Cruz Biotechnology, Inc., USA), the slides were subjected to image acquisitions under a laser scanning microscope (Leica TCS SP5, Leica Microsystems, Exton, PA). The images were then analyzed using the Image-Pro Plus 6.0 (IPP 6.0) software (Media Cybernetics, USA).

Briefly, 5×10⁴ HC dermal fibroblasts were lysed using Laemmli sample buffer (S3401, Sigma) and subsequently 15 µl of total cell lysates were separated by SDS polyacylamide gel and electrophorised onto nitrocellulose membrane. To detect proteins expression antibodies against human TIMP-1 (ab1827, Abcam), TAB1 (C25E9, Cell Signaling), GAPDH (ab9484, Abcam) and secondary HRP- conjugated (Dako) antibody were used. TIMP-1 and MMP-1 (both R&D Systems) protein concentration in culture supernatants were measured by ELISA according to the manufacturer’s protocol. Signal development was performed using HRP/Streptavidin and OPD substrate (Sigma-Aldrich) at room temperature. Fluorescence was measured using a plate reader (Tecan, Sunrise). Samples were run in duplicate and serial dilution was performed in order to fall within the detection limits of the assay (0–80 ng/ml).

Female Wistar rats were used in accordance with the International Ethics Standard for animal experiments. The study was approved by the local Ethics Committee on animal use (protocol n° 732/2012).
The rats were randomly allocated into 2 groups, namely, the Control (CG, n=7) and Stretching (SG, n=8) groups, with the latter being chosen to receive the stretching exercise protocol. The CG was also anesthetized and positioned on the stretching apparatus to be subjected to similar handling and recovery to that of the SG. All rats were subjected to euthanasia after the one-week experimentation period.
After dissection, the soleus muscle was weighed and divided longitudinally into two equal parts for immunohistochemical analysis and extraction of the total RNA according to established methods (15 (link),20 (link)). Immunohistochemical analyses were performed as previously described (15 (link)).
The primary antibodies specific for the immunohistochemical reaction used were as follows: TNF-α: human monoclonal TNF-α (1:50 dilution) (MA-091-5, Imuny Biotechnology, Campinas, SP, BR) and mouse monoclonal TIMP-1 (1:100 dilution) (AB1827, Abcam, Cambridge, MA, USA). A tissue known to express the antigen of interest served as a positive control, whereas experiments performed by omitting the primary antibody constituted the negative control.