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Vector blue alkaline phosphatase substrate kit

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The Vector Blue Alkaline Phosphatase Substrate Kit is a laboratory product designed for use in enzyme-based detection systems. It provides a blue chromogenic substrate for the detection of alkaline phosphatase activity.

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Lab products found in correlation

Biotinylated secondary antibody, by Vector Laboratories (3 mentions) 3 3 diaminobenzidine tetrahydrochloride plus dab, by Thermo Fisher Scientific (3 mentions) Vectastain abc kit, by Vector Laboratories (3 mentions) Ti2e microscope system, by Yokogawa (2 mentions) W1 spinning disk module, by Yokogawa (2 mentions) Y 27632, by Bio-Techne (2 mentions) Accutase, by Merck Group (2 mentions) Nis software, by Nikon (2 mentions) Pcxle hoct3 4 shp53, by Addgene (1 mentions) Basic fibroblast growth factor (bfgf), by Thermo Fisher Scientific (1 mentions) Neon transfection system, by Addgene (1 mentions) β mercaptoethanol, by Thermo Fisher Scientific (1 mentions) Glutamax, by Thermo Fisher Scientific (1 mentions) Af2085, by R&D Systems (1 mentions) Af1924, by R&D Systems (1 mentions) Mab4360, by Merck Group (1 mentions) Mab4304, by Merck Group (1 mentions) Mab4381, by Merck Group (1 mentions) Mab1637, by Merck Group (1 mentions) Gtx101507, by GeneTex (1 mentions)

Spelling variants of this product

Vector Blue (44 citations) Vector Blue Substrate Kit (25 citations) Alkaline phosphatase kit (12 citations) Vector Blue substrate (8 citations) Blue Alkaline Phosphatase Substrate Kit (6 citations) Alkaline Phosphatase (4 citations) Vector blue alkaline phosphatase (Blue AP) substrate kit (4 citations) Vector Blue Alkaline Phosphatase Substrate (3 citations) Alkaline phosphatase substrate (3 citations) Substrate kits (2 citations)

32 protocols using vector blue alkaline phosphatase substrate kit

1

Assessing NANOG Overexpression Effects on Stem Cell Differentiation

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To assess the effects of NANOG WT and mutant overexpression on stem cell differentiation, H9 ESCs with lentiviral transfected NANOG WT or W8A were plated on Cultrex-coated 12-well plates. The cells were initially singularized with Accutase (Sigma-Aldrich, A6964–500ML), then counted and centrifuged. The cell pellets were recovered in StemFlex medium with 10 µM Y-27632 (Tocris, 1254) and 2 µg ml−1 puromycin. Finally, each well was seeded with 40,000 cells. On the next day, the medium (without Y-27632) was changed and NANOG expression was induced with 2 µg ml−1 of DOX for 3–7 days. To check for ESC colonies and undifferentiated cells, alkaline phosphatase activity was assessed using a Vector Blue alkaline phosphatase substrate kit (Vector Laboratories, SK-5300) following the manufacturer’s protocol. The plates were also stained with 0.5% crystal violet solution (protein/nucleic acid stain) in Dulbecco’s phosphate buffered saline (DPBS) to detect all cells. The plates were imaged using a Nikon Ti2E microscope system with a Yokogawa W1 spinning disk module. The large six-well images were derived from stitching of smaller images using the NIS software (Nikon).
Choi K.J., Quan M.D., Qi C., Lee J.H., Tsoi P.S., Zahabiyon M., Bajic A., Hu L., Prasad B.V., Liao S.C., Li W., Ferreon A.C, & Ferreon J.C. (2022). NANOG prion-like assembly mediates DNA bridging to facilitate chromatin reorganization and activation of pluripotency. Nature Cell Biology, 24(5), 737-747.
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2

Generation of Human iPS Cells

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Skin fibroblasts were transfected with pCXLE‐hOct3/4‐shp53, pCXLE‐hSox2‐hKlf4, pCXLE‐hLin28‐hL‐Myc (Addgene, Cambridge, USA) using the Neon Transfection System according to the manufacturer's protocol. After 5 days, the medium was replaced with DMEM/F12 medium supplemented with Glutamax, 20% knockout serum replacer, 1% NAA, 0.1 mmol/l β‐mercaptoethanol (Life Technologies), and 50 ng/ml bFGF (PeproTech, Hamburg, Germany). At day 10, cells were trypsinized and maintained on irradiated mouse embryonic fibroblasts (MEFs) in human iPS cell medium. Colonies were selected based on morphology (Okita et al, 2011; Yu et al, 2011), further passaged, and expanded. As quality control for iPS clones, we performed alkaline phosphatase (Vector Blue Alkaline Phosphatase Substrate Kit; Vector Laboratories, SK5300) and pluripotent markers staining.
Gabriel E., Wason A., Ramani A., Gooi L.M., Keller P., Pozniakovsky A., Poser I., Noack F., Telugu N.S., Calegari F., Šarić T., Hescheler J., Hyman A.A., Gottardo M., Callaini G., Alkuraya F.S, & Gopalakrishnan J. (2016). CPAP promotes timely cilium disassembly to maintain neural progenitor pool. The EMBO Journal, 35(8), 803-819.
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3

Immunohistochemical Tissue Analysis

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Dissected tissues were fixed, immediately after removal, in a 10% buffered formalin solution for a maximum of 24 hr at room temperature before being dehydrated and paraffin-embedded under vacuum conditions. Seven-micrometer sections were deparaffinized in xylene, hydrated in graded alcohols and heated in standard citrate or Tris-EDTA retrieval buffer for 30 min at 95°C. After incubation overnight with the primary antibodies at 4°C, the slides were incubated with biotinylated secondary antibodies (Vector Laboratories Ltd.) for 1 hour at room temperature. Antibody labeling was visualized using the VECTASTAIN ABC kit (Vector Laboratories Ltd.) followed by staining with 3,3’-diaminobenzidine tetrahydrochloride plus (DAB+) according to the manufacturer’s instructions (Thermo Scientific) or using the Vector Blue Alkaline Phosphatase Substrate Kit (Vector Laboratories Ltd.).
di Ronza A., Bajaj L., Sharma J., Sanagasetti D., Lotfi P., Adamski C.J., Collette J., Palmieri M., Amawi A., Popp L., Chang K.T., Meschini M.C., Leung H.C., Segatori L., Simonati A., Sifers R.N., Santorelli F.M, & Sardiello M. (2018). CLN8 is an ER cargo receptor that regulates lysosome biogenesis. Nature cell biology, 20(12), 1370-1377.
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4

Dual IFNγ/IL-10 ELISPOT Assay

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Dual IFNγ/IL-10 ELISPOT (Diaclone, Besancon, France) was performed according to manufacturer’s instructions with a slight modification: visualization of IL-10 was performed using Vector Blue Alkaline Phosphatase substrate kit (Vector Labs, Burlingame, CA, USA) and the A.EL.VIS 4-Plate ELISPOT Reader (A.EL.VIS GmbH, Hannover, Germany) was used. Analysis was performed using ImageJ (version 1.48, NIH, USA) to quantify IFNγ-producing cells (red spots), IL-10-producing cells (blue spots) or dual IFNγ/IL-10-producing cells (purple spots).
Mfarrej B., Tresoldi E., Stabilini A., Paganelli A., Caldara R., Secchi A, & Battaglia M. (2017). Generation of donor-specific Tr1 cells to be used after kidney transplantation and definition of the timing of their in vivo infusion in the presence of immunosuppression. Journal of Translational Medicine, 15, 40.
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5

Pluripotency and Germ Layer Characterization of hc-iPSCs

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The putative hc-iPSCs were fixed with 4% paraformaldehyde (PFA) in DPBS for 20 minutes for immunofluorescent staining, following our routine protocol [22 (link)]. Primary antibodies against OCT4 (1:150, MAB4401, Millipore), SOX2 (1:150, GTX101507, Genetex, Hsinchu, Taiwan), SSEA4 (1:150, MAB4304, Millipore), TRA1-60 (1:200, MAB4360, Millipore) and TRA-1-81 (1:200, MAB4381, Millipore) were used for detecting pluripotency, and those against SOX17, BRACHYURY (1:50. AF1924 and AF2085, R&D Systems Inc., Minneapolis, MN, USA) and β-III-TUBULIN (or TUJ1, 1:200, MAB1637, Millipore) were used for detecting germ layers differentiation. Secondary antibodies include Alexa Fluor goat anti mouse 488 (A11029), goat anti rabbit 488 (A11008), and donkey anti mouse IgM Cy3 (715-165-140, Jackson ImmunoResearch Inc., West Grove, PA, USA). Fixed samples were also subjected to alkaline phosphatase (AP) detection by VECTOR Blue Alkaline Phosphatase Substrate Kit (SK-5300, Vector Laboratories, Burlingame, CA, USA). Teratoma tissues were dissected and immersed in 4% PFA overnight at 4°C and then embedded into wax. Sections were dewaxed, rehydrated and stained with hematoxylin and Eosin (H&E).
Chang W.F., Hwu Y.M., Xu J., Lin C.J., Wang S.W., Cheng A.S., Lu J., Lu C.H, & Sung L.Y. (2016). Derivation of Patient Specific Pluripotent Stem Cells Using Clinically Discarded Cumulus Cells. PLoS ONE, 11(11), e0165715.
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6

Alkaline Phosphatase Staining Protocol

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Alkaline phosphatase (ALP) staining was conducted following the manufacturer’s instructions using the VECTOR Blue Alkaline Phosphatase Substrate Kit (Vector Laboratories).
Zhou J., Cui B., Wang X., Wang H., Zheng J., Guo F., Sun Y., Fan H., Shen J., Su J., Wang J., Zhao H., Tang Y., Gong T., Sun N, & Liang P. (2023). Overexpression of KCNJ2 enhances maturation of human-induced pluripotent stem cell-derived cardiomyocytes. Stem Cell Research & Therapy, 14, 92.
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7

Alkaline Phosphatase Staining for Mineralization

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To measure regions of active mineralization, alkaline phosphatase (AP) staining was performed using the Vector blue alkaline phosphatase substrate kit (Vector Labs) according to manufacturer protocols. The sections were incubated in the substrate solution for 15 minutes. The AP signal was imaged using a Cy5 filter.
Dyment N.A., Breidenbach A.P., Schwartz A.G., Russell R.P., Aschbacher-Smith L., Liu H., Hagiwara Y., Jiang R., Thomopoulos S., Butler D.L, & Rowe D.W. (2015). GDF5 PROGENITORS GIVE RISE TO FIBROCARTILAGE CELLS THAT MINERALIZE VIA HEDGEHOG SIGNALING TO FORM THE ZONAL ENTHESIS. Developmental biology, 405(1), 96-107.
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8

Alkaline Phosphatase Staining Protocol

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Alkaline phosphatase (AP) staining was performed using the Vector blue alkaline phosphatase substrate kit (Vector Labs) according to manufacturer protocols. The sections were incubated in the substrate solution for 15 minutes. The AP signal was imaged using a Cy5 filter (Ex: 640/30 and Em: 690/50).
Hagiwara Y., Dyrna F., Kuntz A.F., Adams D.J, & Dyment N.A. (2019). Cells from a GDF5 origin produce zonal tendon-to-bone attachments following anterior cruciate ligament reconstruction. Annals of the New York Academy of Sciences, 1460(1), 57-67.
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9

Bone and Cartilage Histological Analysis

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Following euthanasia, hindlimbs were harvested and fixed in formalin for 2 days, transferred to 30% sucrose overnight, and embedded in OCT. Tape-stabilized, frozen mineralized sagittal sections35 (link),38 ,39 (link) of the knee were collected and each section was subjected to three rounds of imaging on the Zeiss Axio Scan.Z1 digital slide scanner including 1) fluorescent reporters, mineralization label, and polarized light, 2) alkaline phosphatase (AP) fluorescent staining (Vector Blue Alkaline Phosphatase Substrate Kit, Vector Laboratories) with Hoechst 33342 counterstain, and 3) 0.025% toluidine blue (TB) or hematoxylin and eosin (aqueous) staining. Sections were decalcified prior to alkaline phosphatase staining. The sections displayed in figure 5 were stained with toluidine blue, imaged, then the stain was removed with Immunocal (Statlab), stained for H&E, and finally imaged again such that 4 rounds of imaging occurred. Layered composite images of all imaging rounds were assembled and aligned in image editing software.
Additional Cre-negative double transgenic mice were stained with a mouse anti-αSMA Cy3-conjugated antibody (Millipore Sigma, clone 1A4, 1:200) and an anti-endomucin antibody (Santa Cruz, V.7C7, 1:50) with goat anti-rat Alexa Fluor 647-conjugated secondary antibody (ThermoFisher Scientific, 1:200).
Kamalitdinov T.B., Fujino K., Shetye S.S., Jiang X., Ye Y., Rodriguez A.B., Kuntz A.F., Zgonis M.H, & Dyment N.A. (2019). Amplifying bone marrow progenitors expressing alpha smooth muscle actin produce zonal insertion sites during tendon-to-bone repair. Journal of orthopaedic research : official publication of the Orthopaedic Research Society, 38(1), 105-116.
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10

In Situ Expression Analysis of Limb Genes

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In situ hybridization of whole embryonic limbs to detect Gdf5 and Sox9 was carried out as described (Wilkinson 1992 ). An acetylation step prior to hybridization was necessary for Gdf5, but not for Sox9. Plasmid containing Sox9 (cDNA clone of nt, 116–856; NM_011448) or Gdf5 (cDNA clone of bp 1321–1871; NM_008109), generously provided by Dr. Eiki Koyama, was transcribed into anti-sense and sense probes using digoxygenin-labeled dNTPs (Fisher). Hybridized samples were incubated with a 1:2000 dilution of alkaline phosphatase-conjugated anti-DIG antibody (Cell Signaling) and developed using an alkaline phosphatase chromogenic substrate (BM Purple, Sigma-Aldrich).
Whole mount in situ hybridization using synthesized digoxygenin labeled LNA probe for murine Acvr1 (Exiqon) followed the recommended Exiqon protocol (Sweetman et al 2006 (link), 2008 (link)). Digoxygenin was detected using Vector Blue Alkaline Phosphatase Substrate Kit (SK-5300, Vector Laboratories).
Samples were imaged using an M250C Leica stereomicroscope fitted with a Leica DFC450 C camera.
Towler O.W., Peck S.H., Kaplan F.S, & Shore E.M. (2020). Dysregulated BMP signaling through ACVR1 impairs digit joint development in fibrodysplasia ossificans progressiva (FOP). Developmental biology, 470, 136-146.
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