Immunohistochemical Analysis of c-Met Expression

IHC procedures were completed as previously described [36 (link)]. For antigen retrieval, slides were incubated in 1x citrate buffer (pH 6.0, DBS, Pleasanton, CA) at 100°C in a water bath for 10 min. After peroxidase and protein blocking and treatments, the tissue sections were incubated with the anti-human c-Met antibody (Ab) (2μg/mL, rabbit polyclonal Ab, Santa Cluz Biotechnology, Dallas, TX) at room temperature in a humid chamber for 1 hr. The sections were then visualized using the CSAII kit (Dako, Carpinteria, CA) and Vector VIP Peroxidase (HRP) Substrate Kit (Vector Laboratories, Burlingame, CA) according to the manufacturers' instructions. The sections were counterstained with hematoxylin. Photographs of the IHC staining were taken for analysis using the Nikon Eclipse Ti microscope and NIS elements software (Nikon, Melville, NY). IHC staining intensity values of specimens on slides without the primary Ab were subtracted from the corresponding specimen with the primary Ab to exclude the influence of pigmentation and background staining.