Immunohistochemical Analysis of p53 in ESCC Tissue Microarray

An ESCC TMA, containing a total of 118 formalin-fixed paraffin-embedded tissue samples, was constructed according to a previously described method (10 (link)). IHC was also performed according to a previously described method (11 (link)). Briefly, the sections were deparaffinized in xylene and rehydrated through a gradient concentration of alcohol. The endogenous peroxidase activity was inactivated, non-specific staining was blocked by 5% normal goat serum and all sections were incubated with anti-p53 antibody (1:100; Abmart Inc.) overnight at 4°C. The slides were incubated with biotin-labeled goat anti-rabbit immunoglobulin G and further incubated with streptavidin peroxidase solution (SABC kit, Boster Biological Technology, Ltd., Wuhan, China). The staining was visualized by reaction with 3, 3′-di-aminobenzidine (Boster Biological Technology, Ltd.) in phosphate-buffered saline [PBS; Dycent Biotech (Shanghai) Co. Ltd., Shanghai, China] with 0.05% H₂O₂ for 5 min at room temperature. Control staining was performed by staining the same TMA (duplicate) with PBS rather than anti-p53 and no immunostaining was observed. The slides were counter-stained with hematoxylin, washed in double-distilled H₂O and mounted with resinous mounting medium. The TMA were scored separately by two pathologists who had no prior knowledge of the clinicopathological status of the specimens on the TMA.