Histochemical and biochemical analysis of collagen content in formalin-fixed, paraffin embedded colonic samples: Laboratory protocol for analysis of collagen content in paraffin-embedded tissues

Nicholas Baidoo, Gareth J Sanger, Abi Belai

Research output: Contribution to journalArticlepeer-review


Collagen is the most abundant structural protein and extracellular matrix component in mammals. In the human colon, collagen fibres reside in all the major functional 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) staining. Quantitation estimation of collagen content in each sublayers were performed via ImageJ processing. Hydroxyproline content was quantified using a rapid and sensitive assay in sectioned colonic tissue. Either MT or PSR staining followed by morphometric image analysis via ImageJ provided equally appreciable quantitative results. Moreso, analysis of hydroxyproline content in the 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 utilised to accurately assess collagen content in individual functional sublayers of the colonic wall as well as detection of overall hydroxyproline content in FFPE specimens.

Original languageEnglish
Article numberMEX 102416
Publication statusPublished - 16 Oct 2023


  • Intestinal Collagen
  • Hydroxyproline
  • Masson’s Trichrome
  • Picrosirius red
  • human Colon
  • ImageJ
  • Extracellular matrix (ECM)

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