The genomic DNA isolated from mouse tails was digested by Bsu 36I or Mfe I at 37 °C for 14–16 h according to the manufacturer’s instructions (NEB, Beijing, China). The digested DNA was purified by ethanol precipitation, separated by agarose gel electrophoresis, transferred to an Immobicon-Ny+ membrane (Catalog #: NG0312; GE Healthcare, Beijing, China) and fixed at 120 °C for 30 min. The 5′ Probe-Bsu36I and 3′ Probe-MfeI were synthesized according to the instructions of the PCR DIG probe synthesis Kit (Roche, Shanghai, China) and hybridized with the membrane. After hybridization, the membrane was incubated for 30 min in blocking solution and thereafter in anti-DIG-AP antibody solution (Roche, Shanghai, China) for 30 min. The membrane was subsequently exposed to 1 mL CSPD ready-to-use solution (Roche, Shanghai, China) for 5–20 min and photographs were taken using an imaging system.
Anti dig ap antibody solution
Manufactured by Roche
Sourced in China
The Anti-DIG-AP antibody solution is a laboratory reagent used in various bioanalytical techniques. It is a conjugate of an anti-digoxigenin (DIG) antibody and alkaline phosphatase (AP), which serves as a reporter enzyme. This product is commonly used in applications such as immunoassays, Southern blotting, and in situ hybridization to detect and visualize the presence of DIG-labeled targets.
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2 protocols using anti dig ap antibody solution
1
Southern Blot Analysis of Mouse Genomic DNA
2
miRNA-210 Expression in Brain Tissues
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The expression of miRNA-210 in brain tissues was determined using miRCURY LNA miRNA ISH optimization Kits (Qiagen), including ISH buffer, controls, and double digoxigenin (DIG)-labeled miR-210 detection probe, following the manufacturer’s instructions. Briefly, after Proteinase K incubation, 10-μm-thick frozen section slides were incubated with 50 µL of a diluted solution (40 nM) of LNA miR-210 probe at 55 °C in a humidified chamber for 1 h. Following hybridization, the slides were washed with 5×, 1×, and 0.2× SSC (5 min each wash) at 55 °C. The slides were then incubated for 15 min in a blocking solution (0.1% Tween-20, 2% sheep serum, and 1% BSA in PBS) followed by incubation for 1 h at room temperature in an anti-DIG-AP antibody solution (1:500, Roche). The slides were washed 3 times in PBS-T and then incubated in freshly prepared AP substrate solution (Roche) for 2 h at 30 °C. After washing with water, nuclei were counterstained with Nuclear Fast Red solution (Vector Laboratories). The slides were then dehydrated and mounted using mounting medium (Sigma) for microscopy.
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