The streptavidin-biotin method was used for mutated p53 immunostaining. Antigen retrieval was performed by autoclaving the tissues in pH 6.0 citrate buffer at 121˚C for 15 min.
Endogenous peroxidase activity was quenched using the same method used for PHLDA1-IHC. The sections were sequentially incubated with a polyclonal antihuman p53 antibody (1:200; NCL-L-p53-DO7; Leica Biosystems) for 60 min at room temperature, MULTI for 30 min at room temperature, DAB for color development, and 1% Mayer's hematoxylin for the counterstain. Then, the sections were dehydrated by immersion in a series of alcohol solutions and xylene according to the method used for PHLDA1-IHC. As Shigaki et al (15 (link)) reported, the p53 staining pattern in the nucleus was characterized as sporadic, mosaic, nested, and diffuse. Nested or diffuse patterns were considered to represent p53-IHC positivity regardless of the intensity, whereas the sporadic and mosaic patterns were considered to represent p53-IHC negativity.
Endogenous peroxidase activity was quenched using the same method used for PHLDA1-IHC. The sections were sequentially incubated with a polyclonal antihuman p53 antibody (1:200; NCL-L-p53-DO7; Leica Biosystems) for 60 min at room temperature, MULTI for 30 min at room temperature, DAB for color development, and 1% Mayer's hematoxylin for the counterstain. Then, the sections were dehydrated by immersion in a series of alcohol solutions and xylene according to the method used for PHLDA1-IHC. As Shigaki et al (15 (link)) reported, the p53 staining pattern in the nucleus was characterized as sporadic, mosaic, nested, and diffuse. Nested or diffuse patterns were considered to represent p53-IHC positivity regardless of the intensity, whereas the sporadic and mosaic patterns were considered to represent p53-IHC negativity.