Sigmafast bcip nbt tablet

Western blot was performed according to standard practice and using 12% Precise Tris-Glycine Gels (Thermo Scientific) and Trans-Blot Turbo Mini PVDF Transfer Packs (Bio-Rad). Mouse anti-Renilla luciferase antibody, clone 5B11.2 (Millipore) was used, in 1:1000 dilution, to detect the rLuc transgenic antigen. Donkey anti-mouse IgG conjugated to alkaline phosphatase and SIGMAFAST BCIP/NBT tablet (Sigma-Aldrich) were used to develop the immune reaction. The molecular weights of proteins were compared to the ColorPlus Protein Molecular Weight Markers (New England Biolabs). For H1HA blots, rabbit anti H1HA (diluted 1:500, Sigma: SAB3500059) was used as primary antibody with HRP-conjugate anti-rabbit (diluted 1:5000, Alpha diagnostics, Cat# 20120) as a secondary antibody. For NP-M1 blots, the primary antibody was mouse anti M1 (diluted 1:250, Abcam, Cat# 22396) and the secondary antibody was HRP-conjugate anti-mouse (diluted 1:2500, Alpha diagnostics). Primary rabbit anti-Actin (diluted 1:2500, Sigma: A2066) with secondary anti-rabbit-HRP (diluted 1:5000, Alpha diagnostics: Cat# 20120) were used for Actin blots.

Alkaline phosphatase activity was tested following a protocol for evaluating osteogenic differentiation of MSCs (PromoCell, Heidelberg, Germany). In brief, the cells were rinsed with PBS twice and fixed in 10% neutral buffered formalin (Sigma-Aldrich, MO, United States) for 60 s. Then the cells were washed with washing buffer containing 0.05% Tween 20 (Sigma-Aldrich, MO, United States) in PBS twice (Mediatech Inc., VA, United States), and 1 ml substrate solution [10 ml distilled water contained one SigmaFast™ BCIP/NBT tablet (Sigma-Aldrich, MO, United States)] was added to the wells. The cells were incubated at room temperature for 5–10 min based on color development. The reaction was stopped by rinsing the wells with PBS.

AP staining was performed using a SIGMAFAST BCIP/NBT tablet (Sigma) following the manufacturer's instructions. Briefly, cells were washed twice with PBS and stained with SIGMAFAST BCIP/NBT substrate solution in the dark for 5-10 min. Then, the substrate solution was removed when sufficient color had developed 22 (link). After staining, the cells were washed with PBS, and bright-field images were taken using an inverted fluorescence microscope (TE2000U, Nikon).

Polyclonal antibodies against the native olive β-glucosidase protein purified from olive mesocarp according to the method described by Romero-Segura et al. (2009) (link) were prepared in rabbit by Production and Animal Experimentation General Service of the University of Seville.
For Western blotting, 5–6 μg of protein samples were separated by SDS-PAGE as described above and electro-blotted onto nitrocellulose membrane using the Mini Trans-Blot^® system (Bio-Rad). Bound anti-β-glucosidase primary antibody was detected using an anti-rabbit alkaline phosphatase-conjugated secondary antibody (Sigma–Aldrich, United States). When a mouse monoclonal anti-6xHis antibody (GE Healthcare) was used as primary antibody, an anti-mouse alkaline phosphatase-conjugated antibody (Invitrogen) was employed as secondary antibody. To detect alkaline phosphatase activity after antibodies incubation, nitrocellulose membrane was submerged in a solution obtained by dissolving a SIGMAFAST^TM BCIP^®/NBT tablet (Sigma–Aldrich) in 10 ml of distilled water.

To determine the effects of the CSCs on odontoblastic differentiation, cells were cultured with the tested materials in odontoblastic medium (OM) for 7 days. SHED at a density of 5000 cells/well were seeded on a 48-well plate overnight, and then the medium was changed to odontoblastic differentiation medium (αMEM, 10% FBS, 1% PS, 10 mM transforming growth factor-β1) with different concentrations of material extract dilutions (3.125%, 6.25%, 12.5%, 25%) in 50 µM to 200 µM OM with 100 nM dexamethasone (Sigma) and 50 µg/mL ascorbic acid at 37 °C in 5% CO₂. Differentiated SHED without material extract were used as a positive control. The medium was refreshed every 3 days for 1 week. ALP was measured to determine the effectiveness of differentiation. On Day 7, ALP staining was performed with a staining kit (SIGMAFASTTM BCIP^®/NBT tablet, Sigma-Aldrich, St. Louis, MO, USA). On Day 7, differentiated cells were fixed with 4% paraformaldehyde (Tech & Innovation) at 37 °C for 15 min before being stained with ALP solution dissolved in DW for 1 h in the dark at 37 °C. Light microscopy (Olympus IX71, Shinjuku, Tokyo, Japan) was used to obtain optical images to measure the ALP activity after staining and washing five times with distilled water. Quantification was performed using ImageJ software Java 8 (Bethesda, MD, USA). The extracts were prepared according to ISO 10093-5:2009 [19 ].

H2DCF-DA (2′,7′-dichlorodihydrofluorescein diacetate), Juglone (5-hydroxy-1,4-naphthoquinone), MGDG, quercetin, secondary anti-mouse IgG alkaline phosphatase conjugate and SigmaFast™ BCIP^®/NBT tablets were purchased from Sigma-Aldrich (St. Louis, MO, USA). Bradford Reagent was purchased from Biorad (Mississauga, ON, Canada), secondary alkaline phosphatase conjugate and amyloid antibody 6E10 was purchased from Covance (Montreal, QC, Canada). G. biloba extract (EGb761) was a kind gift from Dr. Willmar Schwabe Pharmaceuticals, Germany.

HeLa cell lysates (15 μg), raw and solubilised E. coli membranes, affinity and size exclusion chromatography fractions, or purified pore proteins (2.2 ng) were separated on NuPAGE Novex 4–12% BisTris SDS gels (Life Technologies) and where indicated, stained with Instant Blue coomassie stain (Expedeon).
For Western blotting, proteins were transferred to PVDF (BioRad or iBlot, Life Technologies) membranes according to standard procedures. The primary antibodies used were anti-GFP (rabbit polyclonal α-GFP, A11122 Life Technologies, 1 in 1000) and anti-TPC2 (rabbit polyclonal α-TPC2, Eurogentec custom antibody, 1 in 1000)24 (link). Blots were developed using a secondary antibody (goat α-rabbit IgG/horseradish peroxidase (HRP) conjugate, 1706515 BioRad, 1 in 2000) and the ECL Prime Western Blotting System (GE Healthcare). All antibodies were incubated for 1 hr at room temperature. His-tagged proteins were detected using a monoclonal α-poly-histidine/alkaline phosphatase conjugate antibody (mouse, A5588 Sigma, 1 in 2000, 2 hrs at room temperature). Blots were developed using SIGMAFAST BCIP/NBT tablets (Sigma), as per the manufacturer’s instructions.