Leukocyte alp kit

A leukocyte ALP kit (Sigma-Aldrich) was used for ALP staining according to the recommended protocol. Cells were counterstained with 0.05% neutral red (Sigma). They were then fixed by immersion in fixative solution (citrate-acetone-formaldehyde) for 30 s, gently rinsed with water and put in an alkaline-dye mixture (sodium nitrite solution, FRV-Alkaline solution, deionized water, Naphthol AS-BI Alkaline solution) for 15 min. The samples were then rinsed for 2 min in deionized water and counterstained for 2 min with Hematoxylin solution Gill N.3, rinsed in tap water and air dried.

ALP staining was performed using Leukocyte ALP kit (Sigma-Aldrich, MO, USA). MC3T3-E1 cells were plated at a density of 5 × 10⁴ cells/well in 24-well plates with 8 mg of DBBM with/without 8 µL of FGF-2. The samples were rinsed twice with PBS and fixed with 4% paraformaldehyde for 20 min after 7 days. An alkaline dye solution was prepared by mixing sodium nitrite solution, fast red violet alkaline solution, deionized water, and naphthol AS-BI alkaline solution. The solution was added to the samples and incubated at room temperature for 15 min. After incubation, the samples were rinsed in deionized water.
The extracellular matrix mineralization was evaluated by Alizarin Red Staining (Alizarin Red S; Sigma-Aldrich) as described previously [37 (link)]. After 14 days of seeding MC3T3-E1 cells, the samples were rinsed twice with PBS and fixed with 96% ethanol for 15 min and stained with 0.2% Alizarin Red Solution in water (pH 6.4) at room temperature for 1 h.
For ALP and Alizarin Red Staining, samples were also incubated without cells and served as “no cell” controls. Images were captured on a microscope camera (Zeiss Stemi 508 with AXIOCAM ERc 5s camera; Carl Zeiss) and imported onto Image J. Color thresholding was used to determine the percent stained values for each field view, according to the method by Fujioka-Kobayashi et al. [37 (link)].