1 step nbt bcip substrate solution

Cells were seeded in the 24-well plate and cultured with the osteogenic medium; α-MEM (Thermo Fisher Scientific) supplemented with 10% certified fetal bovine serum (FBS, Thermo Fisher Scientific), 1% antibiotic and antimycotic solution (A/A, Thermo Fisher Scientific), 10 mM β-glycerol phosphate (Sigma-Aldrich), 50 μM 1 ascorbic acid (Sigma-Aldrich), and 100 nM dexamethasone (Sigma Aldrich). Alkaline phosphatase (ALP) staining was performed on day 7 using 1-Step^TM NBT/BCIP Substrate Solution (Thermo Fisher Scientific), and Alizarin Red staining (pH 4.1, Sigma Aldrich) for calcified bone matrix (Gregory et al., 2004 (link)) was performed on day 21. The deposition of Alizarin Red under mineralization conditions suggests that bone matrix has been deposited. Whole-well images were captured using BZ-X810 (KEYENCE), and ALP and Alizarin Red positive areas were measured using QuPath (Bankhead et al., 2017 (link)).

Osteoclast-like cells and osteoblast-like cells were determined as previously described (Miron et al., 2016 (link); Miron and Bosshardt, 2018 (link); Brooks et al., 2019 (link)). Briefly, in the current study, TRAP staining kit (Sigma Aldrich, St. Louis, MO) was used to identify osteoclast-like cells with multi-nuclei brown staining, and were located on the bone perimeter within resorption lacunae (Sato et al., 2015 (link); Lin et al., 2016 (link); Nabeshima et al., 2017 (link)). Using 3 randomly selected views with x200 magnification in each specimen, in a blind manner, two investigators manually counted the number of TRAP staining positive cells, which were finally normalized by the bone area measured using ImageJ software (Nabeshima et al., 2017 (link)).
In different sections, osteoblast-like cells (Sato et al., 2015 (link); Lin et al., 2016 (link); Nabeshima et al., 2017 (link)) were identified using anti-alkaline phosphatase (ALP) antibody (1-Step^TM NBT/BCIP Substrate Solution, Thermo Scientific, Rockford, IL). Using the entire image of each specimen with x100 magnification, the percentage of brown, positively stained area for ALP was quantified based on the entire area of each section measured using ImageJ software (Ueno et al., 2020 (link)). The color threshold of ALP staining positive area was determined by consensus of three of the investigators.

Osteoblast-like and osteoclast-like cells were identified as previously described (Goodman et al., 2013 (link); Sato et al., 2015 (link); Miron et al., 2016 (link); Brooks et al., 2019 (link)). The tartrate resistant acid phosphatase (TRAP) staining kit (Sigma-Aldrich, St. Louis, MO, United States) was used to identify osteoclast-like cells; multi-nucleated TRAP positive cells located on the bone perimeter within the resorption lacunae were defined as osteoclast-like cells. For the detection of osteoblast-like cells, anti-alkaline phosphatase staining (1-stepTM NBT/BCIP Substrate Solution, Thermo Fisher Scientific, Rockford, IL) was used. The TRAP positive cell number of 6 randomly selected areas in each section and the percentage of ALP positive area of the entire area of each section were quantified using Image J software (National Institutes of Health, United States) according to our previous protocol (Utsunomiya et al., 2021a (link)). The color threshold of each parameter was determined by consensus of three of the investigators. Double-blinded quantitative analysis was conducted by two of the investigators.