Combined staining of elastic and collagen fibers for histopathological and differential diagnosis in aorta pathologies

Histochemical techniques are important for diagnosis of connective tissue fibers involved in different aorta pathologies. The aim of this study was to develop a histochemical staining method that identifies both elastic and collagen fibers simultaneously in the same section of aortic wall. Fragments of aorta from deceased dogs were processed according to standard histological technique for paraffin embedding. Sections were stained with orcein with counterstained with either light green or single step Gomori trichrome, and also orcein stain and picro-sirius red. Orcein stain combined with picro-sirius red allowed visualization of both elastic fibers (brown) and collagen fibers (red) with light microscopy using bright field illumination. Moreover, under polarized light microscopy only collagen fibers were detected, appearing as reddish birefringent fibers, confirming that picro-sirius red-stained fibers were collagen fibers. The combination staining by orcein and picro-sirius solutions provided visualization of both collagen and elastic fibers in the same section, facilitating identification of these connective tissue fibers in the aortic wall.