Antisense RNA probes labeled with either digoxigenin-11 rUTP or fluorescein-12 rUTP (Roche) were synthesized as described by Sive et al. (2000) for the following genes: tubb2b (linearized with BamHI, transcribed with T7) (Klein et al., 2002 (link)), sox2 (linearized with EcoRV, transcribed with T7) (Huyck et al., 2015 (link)), and pcna (linearized with XhoI, transcribed with T7) (Huyck et al., 2015 (link)). PCNA in situ hybridization (ISH) was selected based on reports in the literature suggesting that it represents an effective and accurate tool for assessing dynamic changes in proliferation (Muskhelishvili et al., 2003 (link); Kohler et al., 2005 (link)). Whole-mount chromogenic in situ hybridization (ISH) using nitrobluetetrazolium/5-bromo-4-chloro-3-indolyl phosphate (NBT/ BCIP) alkaline phosphatase substrates was performed as described by Sive et al. with minor modifications (Sive et al., 2000 ). Embryos were photographed using an Olympus SZH10 microscope and an Olympus DP71 camera or Nikon SMZ800N microscope and Nikon DSi-R2 camera. Global image adjustments were made using Adobe Photoshop CC to correct brightness, contrast, and color balance.
Digoxigenin 11 rutp
Manufactured by Roche
Digoxigenin-11 rUTP is a modified nucleotide used in molecular biology applications. It serves as a labeling reagent that can be incorporated into RNA during in vitro transcription, enabling the detection and analysis of the synthesized RNA molecules.
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2 protocols using digoxigenin 11 rutp
1
In Situ Hybridization for Proliferation Markers
2
In Situ Hybridization for Proliferation Markers
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Antisense RNA probes labeled with either digoxigenin-11 rUTP or fluorescein-12 rUTP (Roche) were synthesized as described by Sive et al. (2000) for the following genes: tubb2b (linearized with BamHI, transcribed with T7) (Klein et al., 2002 (link)), sox2 (linearized with EcoRV, transcribed with T7) (Huyck et al., 2015 (link)), and pcna (linearized with XhoI, transcribed with T7) (Huyck et al., 2015 (link)). PCNA in situ hybridization (ISH) was selected based on reports in the literature suggesting that it represents an effective and accurate tool for assessing dynamic changes in proliferation (Muskhelishvili et al., 2003 (link); Kohler et al., 2005 (link)). Whole-mount chromogenic in situ hybridization (ISH) using nitrobluetetrazolium/5-bromo-4-chloro-3-indolyl phosphate (NBT/ BCIP) alkaline phosphatase substrates was performed as described by Sive et al. with minor modifications (Sive et al., 2000 ). Embryos were photographed using an Olympus SZH10 microscope and an Olympus DP71 camera or Nikon SMZ800N microscope and Nikon DSi-R2 camera. Global image adjustments were made using Adobe Photoshop CC to correct brightness, contrast, and color balance.
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