Optimization of Picrosirius red staining protocol to determine collagen fiber orientations in vaginal and uterine cervical tissues by Mueller polarized microscopy

André Nazac, Stéphane Bancelin, Benjamin Teig, Bicher Haj Ibrahim, Hervé Fernandez, Marie-Claire Schanne-Klein, Antonello De Martino
Microsc. Res. Tech.. 2015-06-12; 78(8): 723-730
DOI: 10.1002/jemt.22530

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Nazac A(1)(2), Bancelin S(3), Teig B(4), Ibrahim BH(2), Fernandez H(1)(5), Schanne-Klein MC(3), De Martino A(2).

Author information:
(1)Department of Gynecology and Obstetrics, Bicêtre Hospital, Le Kremlin Bicêtre, France.
(2)Laboratoire De Physique Des Interfaces Et Des Couches Minces (LPICM), Ecole ytechnique, CNRS, Palaiseau, 91128, France.
(3)Laboratoire D’optique Et Biosciences, Ecole Polytechnique, CNRS, INSERM U696, Palaiseau, 91128, France.
(4)Department of Anatomopathology, Bicêtre Hospital, Le Kremlin Bicêtre, France.
(5)Paris XI University, Orsay, France.

Polarized microscopy provides unique information on anisotropic samples. In its
most complete implementation, namely Mueller microscopy, this technique is well
suited for the visualization of fibrillar proteins orientations, with collagen in
the first place. However, the intrinsic optical anisotropy of unstained tissues
has to be enhanced by Picrosirius Red (PR) staining to enable Mueller
measurements. In this work, we compared the orientation mapping provided by
Mueller and second harmonic generation (SHG) microscopies on PR stained samples
of vaginal and uterine cervix tissues. SHG is a multiphoton technique that is
highly specific to fibrillar collagen, and was taken as the « gold standard » for
its visualization. We showed that Mueller microscopy can be safely used to
determine collagen orientation in PR stained cervical tissue. In contrast, in
vaginal samples, Mueller microscopy revealed orientations not only of collagen
but also of other anisotropic structures. Thus PR is not fully specific to
collagen, which necessitates comparison to SHG microscopy in every type of
tissue. In addition to this study of PR specificity, we determined the optimal
values of the staining parameters. We found that staining times of 5 min, and
sample thicknesses of 5 µm were sufficient in cervical and vaginal tissues.

© 2015 Wiley Periodicals, Inc.

DOI: 10.1002/jemt.22530
PMID: 26096960 [Indexed for MEDLINE]

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