Fast violet b salt

For ALP staining, cells were fixed and stained with ALP substrate solution [0.1 mg·mL⁻¹ naphthol AS-MX (Sigma-Aldrich) and 0.6 mg·mL⁻¹ fast violet B salt (Sigma-Aldrich) in 0.1 mol·L⁻¹ Tris-HCl (pH 8.5)] for 30 min at 37 °C. Staining was then quantified with ImageJ software (National Institutes of Health, Bethesda, MD) as previously described.^{43 (link)}

Cells were seeded onto a 12-well plate, with 1 × 10⁴ MC3T3-E1 cells per well. OGFs (100 nM dexamethasone, 0.05 mM ascorbic acid 2-phosphate, and 10 mM b-glycerophosphate; Sigma-Aldrich, St. Louis, MO, USA) were added to trigger osteoblast differentiation under different treatments. After the cells had been treated, they were fixed in 60% citrate-buffered acetone and then incubated with a mixture of Fast Violet B Salt and naphthol AS-MX phosphate alkaline solutions (Sigma-Aldrich, St. Louis, MO, USA) for the detection of ALP expression. ALP activity and the total number of cells were measured using the TRACP & ALP assay kit (TakaRa Bio, Kusatasu, Shiga, Japan) and water-soluble tetrazolium salt (WST-1) reagent (Roche, Basel, Switzerland), respectively, in accordance with the manufacturers’ instructions. Cells were fixed with 10% paraformaldehyde after treatment, and then a 2% Alizarin Red S solution (ScienCell, Carlsbad, CA, USA) was used for detecting calcification. Calcium LiquiColor Assay (Stanbio Laboratory, Boeme, TX, USA) and WST-1 reagent (Roche, Basel, Switzerland) were respectively used in accordance with the manufacturers’ instructions to determine the calcium content and number of cells after treatment.

Quantitative determinations of Alp were performed in MSCs lysates by using the LabAssay™ ALP kit (Wako Pure Chemical Industries, Ltd., Chūō-ku, Osaka, Japan) following manufacturer’s instructions. Absorbance at 405 nm was measured on a VICTOR™ ×3 Multilabel Plate Reader. Cell lysates were analyzed for protein content using the Pierce™ BCA Protein Assay Kit (Thermo Scientific™) according to the manufacturer’s protocol, and intracellular Alp levels were normalized for total protein concentration.
For cytochemical staining, MCSs were washed with PBS and fixed in 4% (v/v) PFA for 10 min at RT. After a wash with water, cells were incubated for 30 min in a solution with 0.01% Naphtol AS-MX phosphate (Sigma-Aldrich^®) and 0.03% Fast Violet B salt (Sigma-Aldrich^®), at RT in the dark. Finally, cells were washed with water and air-dried. Images were acquired in a Leica MZ10F stereomicroscope (Leica).

The method used for ALP staining has been described previously [26 (link)]. MSCs transfected with NC siRNA or PUM2 siRNA were fixed in a 2:3 citrate buffer/acetone fixative. The MSCs were stained for ALP using an alkaline staining solution mixed with fast violet B salt (Sigma-Aldrich) in a naphthol AS-MX phosphate alkaline solution (Sigma-Aldrich) for 30 min in the dark. The stained cells were then rinsed with tap water.

For ALP staining, after formalin fixation (10%) and rinsing twice with PBS (pH 7.4), osteoblasts were maintained with ALP substrate solution, naphthol AS-MX (0.1 mg/ml, Sigma-Aldrich, USA), and fast violet B salt (0.6 mg/ml, Sigma-Aldrich, USA) in 0.1 M Tris-HCl (pH 8.5). In addition, ALP activity was assayed by a LabAssay ALP kit (Wako, Japan) after osteoblasts being rinsed twice with PBS and lysed in Mammalian Protein Extraction Reagent (Pierce, USA) per the specification. Besides, protein quantification using the BCA protein assay kit (Pierce, USA) was implemented under the manufacturer's protocol.
For ARS staining, cells were rinsed twice with PBS and then fixed for 10 min in 2% formaldehyde, followed by 30 min of cell staining with 0.2% ARS solution at 37°C. Each well was rinsed 3 times with distilled water and pictured. The ARS red staining indicated calcium deposition.

Alkaline phosphatase activity from cells in mono- and co-cultures in 6-well tissue culture plates was measured by washing in 1 × PBS, fixing cells in 90 % ethanol and applying 600 μl of 4 % (v/v) Naphthol AS-MX phosphate (Sigma) and 0.0024 % fast violet B-salt (Sigma) mixed in distilled water. Cells were incubated at 37 °C for up to 40 minutes, when the reaction was stopped and the images captured and processed using a Zeiss Axiovert 200 inverted microscope, software version 4.7.