Alkaline phosphatase (ALP) activity was analysed by using an ALP substrate kit III (Vector Laboratories, USA) according to the manufacturer's instructions. Immunostaining assays were performed according to the protocol described previously [24 (link)]. Briefly, cells were fixed with 4% paraformaldehyde, permeabilized in 0.3% Triton X-100, blocked in 10% normal goat serum (Vector Laboratories), and then incubated with primary antibodies against OCT4 (1 : 200, Abcam, USA), NANOG (1 : 200, Abcam, USA), and TNNT2 (1 : 500; Abcam, USA) in 4°C overnight and detected by DyLight 488- or DyLight 549-conjugated secondary antibodies. Nuclei were stained with DAPI (Sigma, USA). A Zeiss Axio Observer A1 fluorescence microscope was used for slide observing and image capture.
Alp substrate kit 3
Manufactured by Vector Laboratories
Sourced in United States
The ALP substrate kit III is a set of reagents designed for the detection and quantification of alkaline phosphatase (ALP) activity. The kit includes a substrate solution and a stop solution, which can be used to measure ALP levels in a variety of biological samples. The core function of the kit is to provide a standardized and reliable method for ALP assays.
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2 protocols using alp substrate kit 3
1
Stem Cell Characterization Protocol
2
Alkaline Phosphatase Activity Assay
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The ALP activity was determined with an ALP substrate kit III (Vector Laboratories, Burlingame, CA, USA) as we previously described [36 (link),37 (link)]. Briefly, the hiPSC clones were fixed in 4% paraformaldehyde and incubated with the fresh vector blue substrate solution for 30–45 min according to the manufacture's instruction. The colonies were imaged with a microscope after washing with the PBS.
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