Immunohistochemical Analysis of Tissue Markers

Tissues were collected at the designated times and snap-frozen in optimal cutting temperature (OCT) medium (Sakura Finetek, Japan), and 8-μm sections were prepared and stained with the following antibodies; Rabbit anti-fibronectin (1:100) (Millipore, Temecula, CA, USA), rabbit anti-vimentin (1:100) (Cell Signalling Technology, Danvers, MA, USA), rabbit anti-N-cadherin (1:1000) (Thermo Scientific, Rockford, IL, USA), as described previously^{13 (link),41 (link)}. Stained sections were analysed using fluorescent or bright-field imaging microscopy (Leica, Germany) and ImagePro Plus Capture and Analysis software (Media Cybernetics, Rockville, MD). Vimentin, Fibronectin and N-Cadherin positive areas were quantified in 15 independent fields/section using Image Pro-Plus software^{41 (link),44 (link)}.