Ir750

Protein co-localization was performed using dual immunofluorescence (IF) staining with the same primary antibodies used for IHC staining. Secondary antibodies used were Vectafluor Excel goat anti-mouse 488 (ready-to-use; cat# DK2488, Vector Laboratories, Burlingame, CA, USA) and Alexa Fluor donkey anti-rabbit 594 (1:500; cat# ab150076, Life Technologies, Carlsbad, CA, USA). All stained slides were mounted using Vecta Shield Hardset mounting medium with 4’,6-diamino-2-phenylindole nuclear stain (cat# H-1200, Vector, Abacus DX, Auckland, New Zealand). Negative controls were performed using matched isotype controls for both mouse (ready-to-use; cat# IR750, Dako, Copenhagen, Denmark) and rabbit (ready-to-use; cat# IR600, Dako).

P53 protein expression was analyzed via immunohistochemistry. The immunohistochemical expression of p53 was studied using an anti-TP53 monoclonal antibody (clone DO-7, 1:100 dilution; DAKO, Glostrup, Denmark) processed with the EnVision (DAKO) system. The TP53 antibody labels wild-type protein, with a very short half-life and is present in small amounts in normal cells. The mutant-type p53 protein, which is the mutated form, has a longer half-life and is detected via positive staining due to a point mutation. Tumor sections were examined for p53 immunoreactivity under a microscope at 20× and 40× magnifications. The expression of p53 was considered positive when the proportion of positive cells was greater than 10% (Figure 3A). Negative p53 immunostaining resulted in tumor sections (Figure 3B). The IHC results were validated using positive and negative tissue controls in all series of the immunostained slides. Positive controls were used to validate p53 on ovarian carcinoma; these tissues contained the target antigen at a known and stable expression level. Positive endothelial and lymphocyte cells were considered as internal positive controls. The negative controls were set up as neoplastic tissue slides stained using mouse isotype antibody Ready-to-Use FLEX Negative Control Mouse (cocktail of mouse IgG1, IgG2a, IgG2b, IgG3, and IgM, IR750, DAKO, Glostrup, Denmark).