Ab83076

Paraffin-embedded sections of rat and human prostate tissue were prepared and incubated with anti-C1q (ab171566 for rat, ab11861 for human), C3 (ab11887 for rat, ab129945 for human), MBL (ab23461 for rat and human), FB (ab192577 for rat and human), and C5b-9 (ab83076 for rat and human) primary antibodies (Abcam). Subsequently, the sections were incubated with appropriate biotinylated secondary antibodies. Staining was detected using a streptavidin-biotin kit (Nichirei, Tokyo, Japan.

Protein was extracted from frozen prostate tissues in RIPA Lysis and Extraction buffer (Thermo Fisher Scientific) and subsequently subjected to SDS-PAGE. Rabbit polyclonal anti-C1q (ab171566), C3 (ab11887), and FB (ab192577) antibodies and mouse monoclonal anti-MBL (ab23461) and C5b-9 (ab83076) antibodies were purchased from Abcam (Cambridge, UK) and used in the experiment as primary antibodies. The appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies were used. Protein bands were visualized with ECL^TM Advance Western Detection Reagent (GE Healthcare, Buckinghamshire, UK) and imaged with a ChemiDoc^TM XRS Plus System (BIO-RAD, Hercules, CA, USA). The membranes were re-probed with a rabbit anti-β-actin polyclonal antibody (CS 4967, Cell Signaling Laboratory, Danvers, MA, USA) and an appropriate secondary antibody to verify that protein loading was similar among the samples.