Images were captured using a Leica microscope (Leica DM5500 photomicroscope equipped with a DFC7000 camera, Leica Microsystem Ltd., Wetzlar, Germany) and LASX software version 3.0 (Leica Microsystem Ltd., Wetzlar, Germany). Histological and immunohistochemical-stained slides were imaged at 10× and 40×, and in oil emersion at 100× magnification in bright field. GPX1 and UBB sections that were counterstained with silver nitrate were visualized using a Texas-Red filter (Leica Microsystem Ltd., Wetzlar, Germany) at >560-nm emission wavelength. The excitation wavelength range for the fluorescent red substrate was set in the range of 365–560 nm at 40× magnification.
Collagen fibrils in Sirius red stained sections were visualized using a Leica microscope, equipped with an additional polarized analyzer, detector (11555079, rotatable; Leica Microsystem Ltd., Wetzlar, Germany), and lambda filter (11513907, Leica Microsystem Ltd., Wetzlar, Germany).
Histomorphometry was performed using a modified trainable weka segmentation plugin [92 (link)] from ImageJ software (version 1.52 d, NIH, Bethesda, MD, USA). The count of osteocytes on silver-nitrate-stained slides was measured using the Cell counter plugin of ImageJ.
Collagen fibrils in Sirius red stained sections were visualized using a Leica microscope, equipped with an additional polarized analyzer, detector (11555079, rotatable; Leica Microsystem Ltd., Wetzlar, Germany), and lambda filter (11513907, Leica Microsystem Ltd., Wetzlar, Germany).
Histomorphometry was performed using a modified trainable weka segmentation plugin [92 (link)] from ImageJ software (version 1.52 d, NIH, Bethesda, MD, USA). The count of osteocytes on silver-nitrate-stained slides was measured using the Cell counter plugin of ImageJ.