Texas red filter

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.

The acid secretion function of osteoclasts was determined by Acridine orange (AO) stain as previously described 41 (link). Osteoclasts cultured for 5 days were stained in α-MEM containing 5 μg/mL of Acridine orange (Sigma) for 15 min at 37℃, washed with PBS, and observed under a fluorescence microscope (Leica Texas Red filter) with a 490 nm excitation filter and a 525 nm arrest filter 29 .