Effect of Ischemia and fixation on 5T4 expression in characterized xenografts models.

A- The four control xenografts models representing high (H226), moderate (H1975), low (H2122), and no (H460) 5T4 protein levels, were grown in mice as described in Materials and Methods, resected and subjected to either no ischemia or 6 hours ischemia. Expression levels are indexed to GAPDH as a housekeeping gene. The expression of 5T4 is highest in the H226 cells, and is unaffected by ischemia. H1975 and H2122 cells have reduced expressions of 5T4, which is again unaffected by ischemia. H460 cells appear to have no measurable 5T4 expression as determined by this technique. The expression levels of 5T4 were assessed using qRT-PCR as described in Materials and Methods. B- The four control xenografts models representing high (H226), moderate (H1975), low (H2122), and no (H460) 5T4 protein levels, were grown in mice as described in Materials and Methods, resected and subjected to either no ischemia or 6 hours ischemia. The expression of 5T4 is highest in the H226 cells, and is unaffected by ischemia. H1975 cells have reduced expression of 5T4 as compared to H226 cells, and there does appear to be a slightly significant increase in protein expression under the influence of ischemia. H2122 cells have reduced expressions of 5T4, which is again unaffected by ischemia. H460 cells appear to have no measurable 5T4 expression as determined by this technique. Overall, there was no evidence of a systemic change in 5T4 expression induced by ischemia across the 4 models analyzed. The expression levels of 5T4 were assessed using Western Blot analysis as described in Materials and Methods. Protein levels are indexed to GAPDH as a housekeeping gene. C- The four control xenografts models representing high (H226), moderate (H1975), low (H2122), and no (H460) 5T4 protein levels, were grown in mice as described in Materials and Methods, resected and fixed. The expression levels of 5T4 were then assessed using IHC analysis as described in Materials and Methods. Staining of 5T4 in H226 xenografts is robust, showing strong circumferential staining of the cells in the model. Strong staining is also seen in the H1975 model. Reduced staining is seen in the H2122 xenograft, with fainter staining of the membranes. No overt membrane staining is seen in the H460 xenograft model.