Immunohistochemical Analysis of Acss2 in Colon Cancer

We performed immunohistochemistry (IHC) on formalin-fixed and paraffin-embedded 5 μm sections from 34 paired stage I-IV human colon cancer and benign tissue samples mounted on poly-l-lysine–coated glass slides and collected by the University of Texas Southwestern Medical Center Tissue Management Core. Tumor and benign tissue samples were present on the same slide when undergoing IHC. We baked slides at 56°C for 30 min, deparaffinized in xylene, and then rehydrated through graded alcohols. We quenched endogenous peroxidase activity by incubating slides in 3% hydrogen peroxide (Cat. No. H1009-100ML, Sigma, Saint Louis, MO) in deionized water for 30 min and rinsing in deionized water twice for 5 min. For Acss2 staining, we submerged slides in BD Retrievagen A (pH 6.0) working solution (Cat. No. 550524, BD Biosciences, San Jose, CA) and heated to ~90°C in a microwave oven for 10 min, followed by slow cooling to room temperature (RT) over 60 min. We then rinsed slides twice with deionized water and once with PBS for 10 min. We blocked non-specific binding by a 60 min incubation with 10% goat serum (Cat. No. S-1000, Vector Laboratories, Burlingame, CA) in PBS. Next, we incubated slides overnight at 4°C with a rabbit monoclonal antibody directed against Acss2 (Cat. No. 3658, Cell Signaling Technology, Danvers, MA; 1:150 in 10% goat serum with PBS) and then 2 hr with biotin-conjugated goat anti-rabbit IgG (Cat. No. 111-065-045, Jackson ImmunoResearch Laboratories, Inc., West Grove, PA; 1:500 in 10% goat serum with PBS). After rinsing in PBS, we incubated slides with HRP-conjugated streptavadin (Cat. No. 43–4323, Invitrogen Corporation, Camarillo, CA; 1:750 in 10% goat serum with PBS) for 60 min at RT. After rinsing in PBS, we visualized immunoreactive deposits by incubation with 3′-amino-9-ethylcarbazole (Cat. No. 00–2007, Invitrogen Corporation, Camarillo, CA). Finally, we counterstained slides with hematoxylin QS nuclear counterstain (Cat. No. H-3404, Vector Laboratories, Burlingame, CA) for 2 min and then coverslipped using Aqua-Poly/Mount Coverslipping Medium (Cat. No. 18606–20, Polysciences, Inc., Warrington, PA).