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Fluorescence of Picrosirius Red Multiplexed with Immunohistochemistry for the Quantitative Assessment of Collagen in Tissue Sections

Collagen is an integral part of structural connective tissue throughout every organ. In wounding, tissue remodeling and certain disease states, the synthesis, degradation, thickness and organisation of collagen fibres is hugely relevant to the functionality of the mammalian extracellular matrix. New methods of quantifying collagen characteristics within tissues in relation to molecular markers of cell behavior are therefore needed to further understand this complex relationship.

Picrosirius Red (PSR) is an increasingly popular histological stain used for collagen detection. The staining method relies on the elongated, anionic structure of the Sirius Red dye binding parallel to cationic collagen fibres.

Aside from regular brightfield microscopy, PSR staining enhances the natural birefringence of collagen fibres when viewed under linear polarised light. Fibres are observed as red, orange, yellow or green, and are usefully indicative of collagen type within the sample. However, sample orientation affects PSR hue and signal strength which can be overcome by using circular polarised light. Fluorescent imaging of PSR stained tissue provides a strong red fluorescence signal that is sensitive, specific for collagen and (unlike liner polarised light microscopy) is unaffected by sample orientation.

In this study, Wegner et al. describe a novel technique where PSR is compatible with multiplex immunostaining, which allows visibility and assessment of overall collagen structure along with specifically immunohistochemically labeled regions of the tissue landscape.

Work was performed on genetically modified mouse prostate epithelial cells that over-expressed beta catenin – distinguished only by immunostaining.

Adult C57BL/6J mouse prostates were fixed in 4% paraformaldehyde, processed, mounted in wax and then sectioned to provide 5µm thick sections upon microscope slides.

Prostate sections were stained with PSR. ROIs were selected within the tissue and were imaged using linear polarised light. To assess orientation dependent changes in appearance, the stage was rotated 45 degrees clockwise, and samples were imaged again until the stage was rotated 180 degrees from its original position.

These same samples were then excited and fluorescence red emission was detected between 635 and 685 nm. Images of the same ROIs were taken with the same stage rotation as for the polarised light images.

When viewed under polarised light, sample rotation altered the birefringent appearance of the fibres. This difference in hue was not seen when visualised with fluorescent light where fibre detail and clarity was improved and was standardised despite sample orientation. Imaging with fluorescent light does not however give any indication as to fibre type within the sample.

This problem was overcome with post PSR staining IHC. Coverslips were removed from these slides and were then pretreated for IHC with rehydration followed by heat induced epitope retrieval (HIER). This process removed all PSR from the tissue and consequently prepared the tissue for IHC (allowing potential use of all available fluorescence channels without background PSR staining). Staining for Collagen I and Collagen IV was subsequently performed. Images of these markers within the same ROI were taken. Fluorescence PSR and IHC images were superimposed using Adobe Photoshop software.

Collagen I and IV immunostaining was detected in discrete areas of the mouse prostate tissue and was visualised as a defined fraction of the total collagen content. These stained regions were subsequently able to be quantified using CT-FIRE fibre quantification software and helped to demonstrate that collagen fibre length was significantly greater near beta catenin over-expressing cells than control cells.

This method of fluorescence PSR microscopy coupled with traditional fluorescence immunostaining offers a new opportunity to quantify collagen locally around immunohistochemically labelled regions of interest and allows more detailed study of relationships between molecular markers of cell behavior and the role of collagens in controlling such behavior.


Wegner et al, Fluorescence of Picrosirius Red Multiplexed With Immunohistochemistry for the Quantitative Assessment of Collagen in Tissue Sections. J Histochem Cytochem 2017 Aug; 65(8):479-490



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