Quantifying Collagen in Tissue and Cells

The concentration of collagen in the paraffin tissue sections and cultured fibroblasts was measured using a “Sirius Red/Fast Green Collagen Staining Kit” (Chondrex Inc, Redmond, US) according to the manufacturer’s instructions. Briefly, collagen proteins were stained with Sirius Red, and non-collagenous proteins were stained with Fast Green. Subsequently, the dye was eluted from the samples with dye extraction solution. The absorbance of the solutions was measured at 540 or 605 nm, respectively. The collagen concentration was calculated from the absorbance. The concentration of collagen per tissue section was calculated as the ratio of collagen to total protein45 (link)46 (link). The data from cultured cardiac fibroblasts were normalized as the fold change to the control (control serum without Ang II stimulation).