Diagnosis and staging had been performed in accordance with the modified Dukes classification. Forty 4 individuals had been guys and 24 individuals had been ladies. The imply age was 58 years having a range of 47 to 70 years. The depth of invasion was classified as mucosal and submucosal mem brane layer, muscular layer and serosa layer. Tumors have been classified too, moderately and poorly differentiated adenocarcinomas. Lymph node metastasis happened in 27 sufferers, along with the other 41 individuals had no metastatic lymph nodes. All sufferers have been followed up for survival. None on the patient underwent radiotherapy or chemo therapy just before surgery. Formalin fixed and paraffin embedded surgical tissue samples were collected from the archives of your Department of Pathology, Wuhan General Hospital of Guangzhou Command.
Immunohistochemistry The paraffin embedded colon cancer tissues and corre sponding distal standard mucosal tissues were reduce at 4 um and mounted on glass slides. Then, the slides had been dewaxed in xylene and rehydrated in ethanol, and treated microtubule stabilizer using a so lution of peroxidase blocking reagent to exhaust endogenous peroxidase activity. They had been place in 0. 01 mol L citrate buffer at pH 6. 0 for 15 mi nutes in an 800 W microwave oven then left at area temperature for 20 minutes to expose antigen hidden inside the tissue due to formalin fixation. To inhibit nonspecific antigen antibody reactions probable in immunohistochemi cal staining, protein blocker was used for 5 minutes, and the slides were washed completely with PBS buffer. Then the slides had been incubated overnight with the primary antibodies against MMP 9 at 4 centigrade.
Biotinylated goat anti mouse secondary anti physique was applied for 20 minutes at space temperature, followed by additional washing with buf fer to remove unbound selleck inhibitor antibody. A complicated of avidin with horseradish peroxidase was then applied for 20 mi nutes at area temperature. For color improvement, the slides were stained with three,three diaminobenzidine tetrahy drochloride, then counterstained with hematoxylin. A reddish brown precipitate inside the cytoplasm of cells indicated a constructive re action. In every single immunohistochemistry run, the optimistic section supplied by the reagent firm served as the optimistic manage, and omission with the key antibody served as adverse handle. Immunohistochemistry stained slides were reviewed by two investigators independently blinded to all clinical information. A scoring method was used to describe each intensity of staining along with the percentage of cells stained. The final score was determined by the combined staining score.