All the steps followed during the entire procedure are detailed in Ferran et al. (2015a ,b ). Sense and antisense digoxigenin-UTP-labeled riboprobes for mouse Dlx5, Pax3, Pax6, Shh, Tcf7l2 and Vegfr2 were synthesized according the manufacturer’s suggestions (Roche Diagnostics S.L., Applied Science, Barcelona, Spain) and using specific polymerases (Fermentas, Madrid, Spain). Probe sequence information is provided in Table 1 . Hybridizations were carried out overnight at 72°C. RNA-labeled probes were detected by an alkaline phosphatase-coupled anti-digoxigenin antibody (diluted 1:3.500; Roche Diagnostics, Manheim, Germany), and the compound nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP; Roche Diagnostics, Manheim, Germany) was used as a chromogenic substrate for the alkaline phosphatase reaction.
Nitroblue tetrazolium 5 bromo 4 chloro 3 indolyl phosphate nbt bcip
Manufactured by Roche
Sourced in Germany
Nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP) is a chromogenic substrate used in various biochemical and immunological assays. It is commonly used for the detection and visualization of enzyme activity, particularly alkaline phosphatase, in a variety of applications such as Western blotting, enzyme-linked immunosorbent assays (ELISAs), and histochemical staining.
Automatically generated - may contain errors
5 protocols using nitroblue tetrazolium 5 bromo 4 chloro 3 indolyl phosphate nbt bcip
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In Situ Hybridization Protocols for Gene Expression
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In situ Detection of miRNA-21 Mutant
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miR# was an artificial mutation of mmu-miR-21: mmu-miR-21 (5′-uag cuu auc aga cug augu uga-3′), mutant mmu-miR-21 (miR#) (5′-uag cuu auc aga cug caca aua-3′). Locked nucleic acid (LNA) probes were purchased from Exiqon (Copenhagen, Denmark). Mouse kidneys were fixed in 10% formaldehyde and incubated with 18% sucrose/PBS overnight at 4°C. Kidney sections (15 mm) were incubated with LNA miRNA probes labeled with digoxigenin at 55°C overnight. After wash, kidney sections were incubated with anti-digoxigenin antibody conjugated with alkaline phosphatase at 25°C for 3 h. Nitro-Blue-Tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate (NBT/BCIP) (Roche) was used for color development with substrates.
3
ASC Osteogenic Differentiation Assay
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To assess the differentiation capacity of ASC towards osteoblasts, ASC (passage 1) were seeded in 12-well culture dishes at 10,000 cells/cm2. The cells were then cultured in monolayer in Osteogenesis Differentiation Medium (Gibco). Control cells were cultured in normal culture medium. Both differentiation and control media were changed twice a week. Differentiation was stopped at 12 days by fixating the cells with 4 % formaldehyde at RT for 10 min. Differentiation was demonstrated by staining alkaline phosphatase (ALP). For this, cells were incubated in 0.2 M TRIS-hydrochloride (pH 10), 0.2 M calcium, 0.1 M magnesium chloride solution for 10 min, whereafter a solution containing 0.2 M TRIS-hydrochloride (pH 10), 0.2 M calcium chloride, 0.1 M magnesium chloride solution and nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate (NBT/BCIP) was added (1:100, Roche Diagnostics, Mannheim, Germany) for 15 min. This stains ALP blue. Microscopic images were taken using a Zeiss microscope.
4
In Situ Hybridization of Mouse Brain Genes
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The tissues were processed for in situ hybridization with digoxigenin‐UTP‐labeled antisense riboprobes. Sense and antisense digoxigenin‐labeled riboprobes for mouse Calb1, Calb2, Enc1, Nkx2.2, and Six3 were synthesized following the manufacter's recommendations (Roche Diagnostics S.L., Applied Science, Barcelona, Spain), and applying specific polymerases (Fermentas, Madrid, Spain). Probe sequence information is provided in Table 2 . In situ hybridization (ISH) was performed basically as described by Ferran, Ayad, et al. (2015 ). After hybridization, all sections were washed and incubated in a solution containing alkaline phosphatase‐coupled anti‐digoxigenin antibody (diluted 1:3.500; Roche Diagnostics). Nitroblue tetrazolium/5‐bromo‐4‐chloro‐3‐indolyl phosphate (NBT/BCIP; Roche) solution was then used as chromogenic substrate for the final alkaline phosphatase reaction (Boehringer, Mannheim, Germany). No specific signal was obtained with sense probes (data not shown).
Dlx1, Dlx2, Dlx5, Dlx6, Isl1, Pax6, Ecel1 and Sst expression was analyzed from in situ hybridization images downloaded from the Allen Developing Mouse Brain Atlas (https://developingmouse.brain-map.org ).
Dlx1, Dlx2, Dlx5, Dlx6, Isl1, Pax6, Ecel1 and Sst expression was analyzed from in situ hybridization images downloaded from the Allen Developing Mouse Brain Atlas (
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In situ Hybridization of Mouse Brain Genes
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