Histochemical and biochemical analysis of collagen content in formalin-fixed, paraffin embedded colonic samples

Collagen is the most abundant structural protein and extracellular matrix component in mammals. In the colon, collagen fibres reside in all the major sublayers; namely, the mucosa, submucosa, muscularis externa and the serosa. Methods to quantify collagen content in formalin-fixed, paraffin-embedded (FFPE) stained sections are required and image analysis offers a technique by which the spatial distribution and localisation of collagen fibres can be easily measured. This laboratory protocol was developed from established techniques using FFPE colon. Human colonic samples embedded transversally in paraffin wax were serially sectioned and stained with either Masson's trichrome (MT) or Picrosirius red (PSR). Quantitation estimation of collagen content in each sublayer was performed via ImageJ processing. Hydroxyproline content was quantified using a rapid and sensitive assay in sectioned tissue. Either MT or PSR staining followed by morphometric image analysis via ImageJ provided equally appreciable quantitative results. Moreso, analysis of hydroxyproline content in our samples indicate that this protocol could be useful in retrospective studies for FFPE samples. This laboratory protocol provides a systematic and reproducible method that can be utilized to accurately assess collagen content in individual sublayers of the colonic wall as well as detection of overall hydroxyproline content in FFPE specimens.