Hemi-mandible sections were stained using 0.2% phosphomolybdic acid (Electron Microscopy Sciences). phosphomolybdic acid was added for 3 min and the slides were subsequently rinsed with water. Sirius Red, 0.1% in saturated picric acid was added to the slides for 90 min, followed by two washes in 0.01 N hydrochloric acid, dehydration, and mounting with Permount (Fisher Chemical Permount Mounting Medium).
Stained sagittal sections were imaged under brightfield light (Nikon Eclipse 80i with Osteomeasure) to quantitate the PDL attachment fraction. The PDL attachment fraction was defined as the total length of acellular cementum along the distal root of the first molar in contact with the PDL, which was subsequently normalized to the total length of the acellular cementum. Slides were also imaged under polarized light to visualize collagen fiber orientation (Carl Zeiss AG Axio Observer D1 Inverted Microscope). Polarized images were then evaluated for PDL fiber length, width and angle using CT-FIRE Matlab extension.52 (link)
Stained sagittal sections were imaged under brightfield light (Nikon Eclipse 80i with Osteomeasure) to quantitate the PDL attachment fraction. The PDL attachment fraction was defined as the total length of acellular cementum along the distal root of the first molar in contact with the PDL, which was subsequently normalized to the total length of the acellular cementum. Slides were also imaged under polarized light to visualize collagen fiber orientation (Carl Zeiss AG Axio Observer D1 Inverted Microscope). Polarized images were then evaluated for PDL fiber length, width and angle using CT-FIRE Matlab extension.52 (link)