Tissue microarray construction Paraffin blocks containing areas consisting of invasive colon carcinoma were identified on corresponding inhibitor ARQ197 H E stained sections as previously described. Areas of interest that represented non necrotic invasive front of the adenocarcinoma were identified and marked on the source block. The source block was cored, and a 1 mm core was transferred to the recipient master block using the Beecher Tissue Microarrayer. Three to six cores of tumor were arrayed per specimen. In addition, a core of normal adjacent colonic mucosa was also sampled when present. Immunohistochemistry Immunohistochemistry for each antigen was done on 5 um thick paraffin sections of colon cancer tissue microarray sample described above.
The microarrays were immunohistochemically stained for phosphorylated RKIP and a full length STAT3 antibody using the Ventana Discovery automated system using the DABMAP and CC1 antigen retrieval. Slides were dehydrated, cleared, and mounted. Positive controls consisted of multitumor and normal tissue microarrays generated in our department. Negative controls included replacement of the primary anti body with non reacting antibodies of the same species. Quantitative immunohistochemical analysis The nuclear and the cytoplasmic staining patterns were separately quantified, for both phosphorylated RKIP and STAT3, using a semiquantitative system for evaluation and grading of the immunostaining pattern, successfully applied by us and others. The phosphorylated RKIP staining intensity was scored into four categories, 0 for complete absence of the staining, 1 for weak staining, 2 for moderate, and 3 for strong staining.
The extent of the positively stained cells was also scored into a percentage. Each core was given a score derived from the calculation of grade 1 percentage 100. Score of each case is the average of all the cores of the case. At least three cores were scored per case. The STAT3 staining intensity was scored in the same fashion. The score ranges from 0 to 3. This scoring system takes both intensity and extension into consideration. To convert it into a more understandable quantile format, scores of 0 are graded as 0, scores 0 and 1 are graded as 1, scores 1 and 2 are graded as 2, and scores 2 are graded as 3. All sections were scored independently by SL and were blinded to the clinicopathologic features or clinical outcome.
Statistical analysis Chi square analysis was used to evaluate the association between STAT3 expression and tumor grade and lymphovascular invasion in tumor. All tests were two sided and p values of 0. 05 or less were considered statistically significant. Statistical Axitinib VEGFR1 analyses were done using the JMP 8. 0 statistical program. The vast majority of the cases have a complete set of staining data and clinicopathologic information upon which statistical analysis was performed.
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