Anti igfbp4

To validate the accuracy of iTRAQ results, western blot analysis was performed.
The plasma was diluted ten times by PBS. 30 μL of samples per lane were loaded for 10% Bis-Tris polyacrylamide gels and then transferred onto PVDF membrane (Millipore, Billerica, MA) by electroblotting. The membranes were blocked in TBS 0.5% Tween containing 5% skim milk powder (OXOID, Basingstoke, UK) for 1 h at room temperature and incubated with primary antibody: anti-IGFBP4 (Abcam, Cambridge, UK) overnight at 4 °C. Then the membranes were incubated with peroxidase-conjugated secondary antibodie (Bio-Rad, Hercules, USA) for 1 h at room temperature. The bands were visualized by using Millipore’s enhanced chemiluminescence (ECL) with the Amersham Imager 600 detection system (GE, Boston, USA). The western blot results represent at least 2 independent biological replicates.

A total of 100 primary bladder cancer samples from 2013 to 2019 were enrolled in this study from the Department of Urology, Foshan Maternal and Child Health Hospital, Southern Medical University. The clinicopathological information of these patients is summarized in Table 1. The study was approved by the Ethics Committee of Southern Medical University and informed consent was obtained from all patients. All resected bladder tissues were fixed in 10% formaldehyde for 48 hours and embedded in paraffin. Then paraffin-embedded tissues were cut into 5um thick sections. Slides were deparaffinized with xylene and rehydrated with graded alcohol for pretreatment. Antigen retrieval was performed by microwave heating with citric acid buffer. To block endogenous peroxide, slides were separated with 3% H₂O₂ for 10 min in a wet box. Then, slides were with primary antibodies anti-IGFBP4 (Abcam, Cambridge, MA) at a dilution of 1: 100 overnight at 4 ° C in a humidified box. After washing with phosphate-buffered saline, slides were maintained with a secondary HRP-labeled anti-rabbit antibody (1:50, Beyotime, Shanghai, China) for 30 min at room temperature. Tissue sections were stained with 3,3'-diaminobenzidine (DAB) and counterstained with hematoxylin. Images were acquired with a Nikon microscope camera (Nikon Americas Inc, NY).