Uninjured mice were sacrificed at the age of 10 weeks. In DMM or sham surgery groups, animals were sacrificed at either 2 weeks or 8 weeks postoperatively. Specimens were fixed in 4% paraformaldehyde for 24 hours, decalcified with 14% ethylenediaminetetraacetic acid (EDTA) for 14 days, and embedded in the optimum cutting temperature (OCT) compound. Sections were prepared at 6 μm thickness with a Cryofilm type 3c (SECTION-LAB, Japan).
For immunofluorescent immunohistochemistry, sections were washed in 1× PBS, blocked in 5% normal goat serum (S-1000, Vector Labs, CA, USA) for 30 min, and incubated with primary antibodies specific for CD31 (1:50, ab28364, Abcam, MA, USA), α-SMA (1:400, ab7817, Abcam), ED-A fibronectin (1:400, F6140, Sigma Aldrich, MO, USA) or TGF-β1(1:400, ab92486, Abcam) at 4 °C overnight. Sections were then incubated with Alexa Fluor® 647-conjugated secondary antibodies (1:200, ab150083 or ab150119, Abcam), and mounted with mounting medium containing DAPI (H-1500, Vector Labs). Bright field images were obtained on a Leica DM 6B microscope (Leica Biosystems, Germany). Immunofluorescent images were acquired on a LSM 780 FCS confocal microscope (Carl Zeiss, Germany).
For immunofluorescent immunohistochemistry, sections were washed in 1× PBS, blocked in 5% normal goat serum (S-1000, Vector Labs, CA, USA) for 30 min, and incubated with primary antibodies specific for CD31 (1:50, ab28364, Abcam, MA, USA), α-SMA (1:400, ab7817, Abcam), ED-A fibronectin (1:400, F6140, Sigma Aldrich, MO, USA) or TGF-β1(1:400, ab92486, Abcam) at 4 °C overnight. Sections were then incubated with Alexa Fluor® 647-conjugated secondary antibodies (1:200, ab150083 or ab150119, Abcam), and mounted with mounting medium containing DAPI (H-1500, Vector Labs). Bright field images were obtained on a Leica DM 6B microscope (Leica Biosystems, Germany). Immunofluorescent images were acquired on a LSM 780 FCS confocal microscope (Carl Zeiss, Germany).