Punch biopsies were fixed in 10% formalin, embedded in paraffin, and sectioned to a thickness of 6 μm on a microtome (Leica, Germany). Slides were deparaffinized using xylene and rehydrated through an ethanol gradient. Staining was then performed by Masson’s trichrome or immunofluorescence using previously described protocols [28 , 31 ]. For immunofluorescence, primary antibodies included monoclonal anti-mouse collagen type I (Abcam, Cambridge, UK) and monoclonal anti-mouse collagen type IIII (Abcam, Cambridge, UK) at a 1:500 dilution. A polyclonal goat anti-mouse IgG-Cy3 (Abcam, Cambridge, UK) conjugated secondary antibody at a dilution of 1:100 was also used.
Image J (v1.48, NIH) imaging software was used to analyze Masson’s trichrome digital images and quantify the amount of collagen per high powered field. A Zeiss Axioimager microscope equipped with both a color and monochrome camera with fluorescent filters was used to view slides (Carl Zeiss, Oberkochen, Germany). Zeiss Zen Pro 2012 software (Carl Zeiss, Oberkochen, Germany) was then used to capture digital images and quantify staining intensity of collagen types I and III.
Image J (v1.48, NIH) imaging software was used to analyze Masson’s trichrome digital images and quantify the amount of collagen per high powered field. A Zeiss Axioimager microscope equipped with both a color and monochrome camera with fluorescent filters was used to view slides (Carl Zeiss, Oberkochen, Germany). Zeiss Zen Pro 2012 software (Carl Zeiss, Oberkochen, Germany) was then used to capture digital images and quantify staining intensity of collagen types I and III.