The gene-transfected DO-11.10 cells and silk solutions from W/cs1, S01, and WS19 strains were treated with 2 × SDS sample buffer, separated using 12% SDS-PAGE, and transferred to polyvinylidene difluoride membranes (Bio-Rad). The blots were blocked with Blocking One (Nacalai Tesque, Kyoto, Japan) for one hour at room temperature and incubated with anti-Myc polyclonal antibody (MBL, code no. 562, Nagoya, Japan), anti-β-actin rabbit MAb (Cell Signaling Technology, code no. #4970, Danvers, MA, USA), and anti-FibL polyclonal antibody (raised against a synthetic peptide representing FibL residues 67–80), followed by alkaline phosphatase-conjugated anti-rabbit immunoglobulins (Igs) (Dako, code no. D0306, Glostrup, Denmark). Immunoreactive proteins were detected using BCIP-NBT Solution Kit for Alkaline Phosphatase Stain (Nacalai Tesque).
Bcip nbt solution kit for alkaline phosphatase stain
Manufactured by Nacalai Tesque
The BCIP-NBT Solution Kit is a reagent used for the detection and visualization of alkaline phosphatase activity in various biological applications, such as immunohistochemistry, in situ hybridization, and enzyme-linked immunosorbent assays (ELISAs). The kit contains a pre-mixed solution of BCIP (5-bromo-4-chloro-3-indolyl phosphate) and NBT (nitro blue tetrazolium) that reacts with the alkaline phosphatase enzyme to produce a dark-blue, insoluble precipitate, allowing for the localization and quantification of the target analyte.
Automatically generated - may contain errors
Lab products found in correlation
Anti β actin rabbit mab, by Cell Signaling Technology (1 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (1 mentions)
Blocking one, by Nacalai Tesque (1 mentions)
Alexa 488, by Thermo Fisher Scientific (1 mentions)
Osteogenic differentiation medium, by PromoCell (1 mentions)
Adipocyte differentiation medium, by Cell Applications (1 mentions)
Oil red o, by Nacalai Tesque (1 mentions)
3 protocols using bcip nbt solution kit for alkaline phosphatase stain
1
Western Blot Analysis of Transfected Cells
2
Cell Sorting and Characterization
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Cells' sorting was done using a MoFlo (DakoCytomation). ALP staining was done using BCIP-NBT solution kit for alkaline phosphatase stain (Nacalai Tesque) according to the manufacturer's instructions. Immunostaining was done using antibody anti-Ki67 proliferation antigen (BD Biosciences), and the primary antibody was visualized using appropriate secondary antibody conjugated with Alexa 488 (Molecular Probes).
3
Osteoblast and Adipocyte Differentiation
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ASCs were differentiated into osteoblasts in osteogenic differentiation medium (PromoCell, Heidelberg, Germany). Alkaline phosphatase of differentiated cells was visualized using the BCIP-NBT Solution Kit for Alkaline Phosphatase Stain (Nacalai), and differentiated into mature adipocytes in adipocyte differentiation medium (Cell Applications Inc.). Lipid accumulation in adipocytes was visualized by Oil Red O (Nacalai) according to the manufacturer’s protocol.
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