A simple method to assess the relative amount of collagen deposition in wounded fetal mouse limbs

A modification of the Sirius red and fast green dye staining technique which binds selectively to collagen and noncollagenous proteins, respectively, has been used to quantify the amount of collagen deposition occurring in wounded fetal mouse limbs. Wounded day 14 and 18 fetal mouse limbs were grown in serum-free organ culture for 1 to 7 days, fixed in formalin, embedded in paraffin, and sectioned. The sections were stained with Sirius red and fast green dyes, and those sections obtained from either wounded or unwounded tissue were identified microscopically. The sections were then scraped off microscopic slides. Dye bound to the tissue sections was then eluted with a mixture of sodium hydroxide and methanol, and the optical densities were determined spectrophotometrically. There was a 98.5% correlation between the absorbance of Sirius red dye (collagen) and fast green dye (noncollagenous proteins) of eluted stain and hydroxyproline and leucine content, respectively, as determined by high-performance liquid chromatography. In addition, there was a greater collagen/protein ratio in wounded compared with unwounded sections of day 18 limbs at 7 days after wounding (p = 0.005). However, no difference in the collagen/protein ratio was detected between wounded and unwounded regions of day 14 limbs at either day 1 or 7 after wounding. These results are consistent with previous histologic observations indicating greater collagen deposition in wounded regions of day 18 compared with day 14 limbs at 7 days after wounding. With the use of this technique, it is now possible to quantify the effects of putative fibrogenic agents on collagen deposition in wounded embryonic tissue.