Cy3 conjugated goat anti mouse igg

The tissue block was cut into 10 μm thick sections and dried on glass slides at room temperature overnight. The sections were washed with PBS for 3 times, fixed with 4% paraformaldehyde for 30 min, and then washed with PBS for another 3 times. For histochemical analysis, the sections were stained with H&E. For immunohistochemical staining, the sections were incubated at room temperature in a humid chamber with 5% goat serum for 30 min. After the serum was carefully removed, the sections were incubated either with mouse anti-collagen type I or rabbit anti-collagen III or mouse anti-MMP-3 antibodies (1:350; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or mouse anti-von Willebrand Factor (vWF) (1:200; Bio-Rad, Raleigh, NC) antibodies at room temperature for 2 hrs. The tissue sections were washed with PBS 3 times, the expression of collagen I, MMP-3, and vWF were determined by Cy3-conjugated goat anti-mouse IgG (1:500; Santa Cruz Biotechnology) and collagen III expression was tested by Cy-3-conjugated goat anti-rabbit IgG (1:500; Santa Cruz Biotechnology) at room temperature for 1 hr, further washed with PBS 3 times, and Hoechst fluorochrome 33342 (1μg/ml; Sigma, St. Louis, MO) was used for staining the nuclei at room temperature for 5 min. Finally, the sections were washed with PBS three times and the stained sections were examined using fluorescence microscopy.