To assess the expression pattern of CADM1-AS1 in HCC, in situ hybridization was performed with double digoxigenin-labeled probes (Exiqon, Vedbaek, Denmark) according to the manufacturer’s instruction. Briefly, HCC tissue samples were sectioned at 5 μm and deparaffinized, then treated with proteinase-K (5 μg/ml) for 2 min at 37 °C. Slides were prehybridizated with the 1 × ISH buffer (Exiqon) and the samples were hybridized with digoxigenin-labeled probes at 50 °C for 1 h. Next, the slides were incubated with anti-digoxigenin antibody (Roche Diagnostics, IN) at 4 °C overnight. The probe sequence for CADM1-AS1 was 5ʹ-TCAGCCATAGTGCATAGCTACT-3′. Staining intensity was scored as 0 (negative), 1 (weak), 2 (medium) and 3 (strong). The staining extent was scored as 0 (10%), 1 (11–25%), 2 (26–50%), 3 (51–75%) and 4 (76–100%). These two subscores were then multiplied to obtain a final staining index. Low CADM1-AS1 expression was defined as a staining index of ≤3, whereas high CADM1-AS1 was >3 as described in our previous study.
Double digoxigenin labeled probes
Manufactured by Qiagen
Sourced in Denmark
Double Digoxigenin-labeled probes are a type of molecular biology tool used for the detection and visualization of specific DNA or RNA sequences within a sample. They consist of a DNA or RNA sequence that is labeled with two digoxigenin molecules, which can be recognized by an anti-digoxigenin antibody conjugated to a reporter molecule, such as a fluorescent dye or an enzyme. These probes can be used in various applications, including in situ hybridization, Northern blotting, and Southern blotting, to identify and localize target sequences within cells or tissues.
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2 protocols using double digoxigenin labeled probes
1
In Situ Hybridization of CADM1-AS1 in HCC
2
In Situ Hybridization of PVT1 in PDA
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To explore the expression pattern of PVT1 in PDA, in situ hybridization was conducted with double digoxigenin-labeled probes (Exiqon, vedbaek, Denmark) according to the manufacturer’s instruction. Briefly, the PDA tissues were sectioned at 4 μm thick and deparaffinized, then treated with proteinase-K (20 μg/ml) for 10 min at 37 °C. Slides were prehybridizated with the 1 × ISH buffer (Exiqon) and then hybridizated with digoxigenin-labeled probes at 45 °C for 1 h. Afterwards, the slides were incubated with anti-digoxigenin antibody (Roche Diagnostics, IN) at 4 °C overnight, and then stained with nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate. The sequences of the probes are as follows: PVT1 probe: 5’-AACAGGGCAGGATCTATGGCAT-3′ and scramble probe: 5’-GTGTAACACGTCTATACGCCCA-3′.
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