Anti dig fab antibody

Full-length p53 sequences derived from a pEGFP-C2 p53 plasmid were used to synthesize the complementary RNA (cRNA) probes for p53. The plasmid was digested with restriction enzymes, and then labeled with Digoxigenin (DIG) by in vitro transcription using the RNA labeling kit (Roche Diagnostics, Indianapolis, IN, USA). Four-micrometer sections obtained from paraformaldehyde-fixed and paraffin-embedded tissue samples were deparaffinised, rehydrated, and incubated with 0.5% acetic anhydride solution to remove nonspecific binding. Hybridization was performed with DIG-sense and -antisense cRNA probes in 20× SSC solution containing 50% formamide at 42 °C overnight. The next day, the slides were washed in the SSC solution at 50 °C and incubated with an anti-DIG Fab antibody (Roche Diagnostics) conjugated to alkaline phosphatase (AP), at 4 °C overnight. To detect alkaline phosphatase, nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP, Roche Diagnostics) was added. Nuclei were counterstained with Nuclear Fast Red (Vector Laboratories, Inc., Burlingame, CA, USA).

An antibody against endothelial differentiation-related factor 1 (EDF1) (BS70093) was obtained from Bio World, Nanjing, China. An antiaromatase antibody (BA3704) was obtained from BOSTER, Wuhan, China. Real-time polymerase chain reaction (PCR) mix (208054) was obtained from QIAGEN, Germany. ANTI-FLAG^®M2 magnetic beads (M8823) were obtained from Sigma, St. Louis, MO, United States. Phusion^® high-fidelity DNA polymerase and endonuclease were obtained from New England Biolab, Ipswich, MA, United States. FuGENE 6 transfection reagent (E2691) was purchased from Promega, Madison, WI, United States. An anti–β-actin antibody (HC201), anti-Flag antibody (HT201), and a CCK8 kit (FC101) were obtained from TransGen Biotech, Beijing, China. The digoxigenin (DIG) RNA labeling mix (11277073910) and anti-DIG-Fab antibody (110932749100) were obtained from Roche, Mannheim, Germany. pLV[shRNA]-EGFP:T2A:Puro-U6 > mEdf1 plasmid was obtained from Vector Builder, Guangzhou, China.