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

The low cost and simplicity of picrosirius red (PSR) staining have driven its popularity for collagen detection in tissue sections. We extended the versatility of this method by using fluorescent imaging to detect the PSR signal and applying automated quantification tools. We also developed the firs...

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Bibliographic Details
Published in:The journal of histochemistry and cytochemistry 2017-08, Vol.65 (8), p.479-490
Main Authors: Wegner, Kyle A., Keikhosravi, Adib, Eliceiri, Kevin W., Vezina, Chad M.
Format: Article
Language:English
Subjects:
Animals
Azo Compounds - chemistry
beta Catenin - genetics
beta Catenin - metabolism
Collagen - analysis
Coloring Agents - chemistry
Epithelial Cells - chemistry
Epithelial Cells - metabolism
Fibrillar Collagens - analysis
Immunohistochemistry - methods
Male
Mice, Inbred C57BL
Optical Imaging
Prostate - chemistry
Prostate - metabolism
Reproducibility of Results
Sensitivity and Specificity
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Summary:The low cost and simplicity of picrosirius red (PSR) staining have driven its popularity for collagen detection in tissue sections. We extended the versatility of this method by using fluorescent imaging to detect the PSR signal and applying automated quantification tools. We also developed the first PSR protocol that is fully compatible with multiplex immunostaining, making it possible to test whether collagen structure differs across immunohistochemically labeled regions of the tissue landscape. We compared our imaging method with two gold standards in collagen imaging, linear polarized light microscopy and second harmonic generation imaging, and found that it is at least as sensitive and robust to changes in sample orientation. As proof of principle, we used a genetic approach to overexpress beta catenin in a patchy subset of mouse prostate epithelial cells distinguished only by immunolabeling. We showed that collagen fiber length is significantly greater near beta catenin overexpressing cells than near control cells. Our fluorescent PSR imaging method is sensitive, reproducible, and offers a new way to guide region of interest selection for quantifying collagen in tissue sections.
ISSN:0022-1554
1551-5044
DOI:10.1369/0022155417718541