Pab19095

A total of 90 LSTs, 30 normal tissues, and 30 TNM stage I colorectal carcinomas were subjected to IHC analysis. Briefly, serial consecutive 4-μm tissue sections were cut for subsequent study. Hematoxylin and eosin (H&E) staining was performed according to standard procedures and used for histological verification. IHC staining was performed using EnVision+ Kit (Dako, Denmark). The slices were separately incubated at 4 °C overnight with primary antibodies against LCN-2 (ab23477, 1: 200; Abcam) and MMP-9 (PAB19095, 1:100; Abnova). Based on histological verification by an experienced pathologist, 90 LST cases were classified into intestinal intraepithelial neoplasia, including Mild, Moderate and Severe in this study. For IHC quantitative analysis, scoring of tissue slides was evaluated in a blinded manner by two investigators. The ratio of positively stained cells to all cells in eight random areas at 200-fold magnification was recorded. The total score was then generated based on the average staining intensity and the average percentage of positive cells as previously described35 (link).

Thirty micrograms of protein samples from LSTs and other groups were separated on a 10% or 12% SDS-PAGE and transferred to nitrocellulose membranes (Millipore). Protein bands were visualized using a conventional enhanced chemiluminescence (ECL) system (GE Healthcare, USA) and quantified with Quantity One (Bio-Rad, USA). Primary antibodies used were anti-LCN-2 antibody (ab23477, 1:500; Abcam), anti-MMP-9 (PAB19095, 1:200; Abnova), and anti-beta tubulin antibody (ab15568, 1:200; Abcam).