Freshly collected hindlimbs of mouse embryos (E16.5) were fixed in 4% (weight/volume) paraformaldehyde and embedded in paraffin. Serial sections (7 μm) were either stained for TRAP activity or used for in situ hybridization. For the syncytin-B gene, three PCR-amplified fragments of 414, 511, and 370 bp, designed in the regions the least conserved between the syncytin-A and -B genes were cloned into pGEM-T Easy (Promega) (Vernochet et al., 2014 (link)). In vitro synthesis of the antisense and sense riboprobes was performed with SP6 or T7 RNA polymerase and digoxigenin 11-UTP (Roche Applied Science) after cDNA template amplification. Sections were processed, hybridized at 42 °C overnight with the pooled riboprobes (1 μg/mL of each riboprobe), and incubated further overnight at 4 °C with alkaline phosphatase-conjugated anti-digoxigenin antibody Fab fragments (Roche Applied Science). Staining was achieved with the nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate phosphatase alkaline substrates as indicated by the manufacturer (Roche Applied Science). To assess the specificity of the syncytin-B probes, they were tested on placenta sections prepared from SynB−/− mice. No staining was observed, indicating that the syncytin-B probe does not cross react with syncytin-A transcripts (data not shown).
Alkaline phosphatase conjugated anti digoxigenin antibody fab fragments
Manufactured by Roche
The Alkaline phosphatase-conjugated anti-digoxigenin antibody Fab fragments are laboratory reagents used to detect the presence of digoxigenin, a small organic molecule. The Fab fragments are derived from antibodies and are conjugated with the enzyme alkaline phosphatase, which can be used to generate a colorimetric or luminescent signal when exposed to appropriate substrates. This product is commonly used in various analytical techniques, such as immunoassays and in situ hybridization, to identify and quantify the presence of digoxigenin-labeled targets.
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2 protocols using alkaline phosphatase conjugated anti digoxigenin antibody fab fragments
1
In Situ Hybridization of Syncytin-B in Mouse
2
In Situ Hybridization for syncytin-B
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Freshly collected muscles were fixed in 4% (wt/vol) paraformaldehyde and embedded in paraffin. Serial sections (7 μm) were used for ISH. For the syncytin-B gene, three PCR-amplified syncytin-B fragments of 414, 511, and 370 bp, respectively (primers listed in S1 Table ) were cloned into pGEM-T Easy (Promega). In vitro synthesis of the antisense and sense riboprobes was performed with SP6 or T7 RNA polymerase and digoxigenin 11-UTP (Roche Applied Science) after cDNA template amplification. Sections were processed, hybridized at 42°C overnight with the pooled riboprobes, and incubated further overnight at 4°C with alkaline phosphatase-conjugated anti-digoxigenin antibody Fab fragments (Roche Applied Science). Staining was achieved with the nitroblue tetrazolium (NT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP) phosphatase alkaline substrates as indicated by the manufacturer (Roche Applied Science).
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