D6h2l

Serial tissue sections of 4-μm thickness, containing the deepest area of the tumor invasion, were depara nized in xylene, rehydrated in graded alcohol, and immersed in 3.0% hydrogen peroxide in methanol for 10 min at room temperature to inhibit endogenous peroxidase activity. For antigen retrieval, the tissue slides for GR, Sgk1, and NDRG1 immunohistochemistry were heated in an autoclave at 121 °C for 5 min in 0.01 M citrate buffer (pH 6.0). After washing three times for 5 min each in phosphate-buffered saline (PBS), the reacted slides were incubated in 1% normal goat serum for 30 min at room temperature to reduce nonspeci c antibody binding and then incubated at 4 °C overnight with rabbit monoclonal antibody against GR (D6H2L, Cell Signaling Technology, Danvers, MA, USA, diluted 1/400), Sgk1 (Y238, Abcam, Cambridge, UK, diluted 1/200), or NDRG1 (EPR5593, Abcam, diluted 1/400). The reacted sections were then washed three times for 5 min each in PBS, incubated with biotinylated anti-rabbit immunoglobulin (Nichirei Biosciences, Inc., Tokyo, Japan), washed three times for 5 min each in PBS, and incubated with peroxidaselabeled streptavidin (Nichirei Biosciences, Inc.) for 30 min at room temperature. Immunoreactivity was visualized with 3,3 ′ -diaminobenzidine, and the slides were counterstained with Mayer's hematoxylin, dehydrated in graded alcohol, and cleared in xylene.

Serial tissue sections of 4-μm thickness, containing the deepest area of the tumor invasion, were depara nized in xylene, rehydrated in graded alcohol, and immersed in 3.0% hydrogen peroxide in methanol for 10 min at room temperature to inhibit endogenous peroxidase activity. For antigen retrieval, the tissue slides for GR, Sgk1, and NDRG1 immunohistochemistry were heated by autoclave at 121°C for 5 min in 0.01 M citrate buffer (pH 6.0). After washing three times for 5 min each in phosphate-buffered saline (PBS), the slides were incubated in 1% normal goat serum for 30 min at room temperature to reduce nonspeci c antibody binding and then incubated at 4°C overnight with rabbit monoclonal antibody against GR (D6H2L, Cell Signaling Technology, Danvers, MA, USA, diluted 1/400), Sgk1 (Y238, Abcam, Cambridge, UK, diluted 1/200), or NDRG1 (EPR5593, Abcam, diluted 1/400). The reacted sections were then washed three times for 5 min each in PBS, incubated with biotinylated anti-rabbit immunoglobulin (Nichirei Biosciences, Inc., Tokyo, Japan), washed three times for 5 min each in PBS, and incubated with peroxidase-labeled streptavidin (Nichirei Biosciences, Inc.) for 30 min at room temperature. Immunoreactivity was visualized with 3,3 ′ -diaminobenzidine, and the slides were counterstained with Mayer's hematoxylin, dehydrated in graded alcohol, and cleared in xylene.

Serial tissue sections of 4-μm thickness, containing the deepest area of the tumor invasion, were deparaffinized in xylene, rehydrated in graded alcohol, and immersed in 3.0% hydrogen peroxide in methanol for 10 min at room temperature to inhibit endogenous peroxidase activity. For antigen retrieval, the tissue slides for GR, Sgk1, and NDRG1 immunohistochemistry were heated in an autoclave at 121 °C for 5 min in 0.01 M citrate buffer (pH 6.0). After washing three times for 5 min each in phosphate-buffered saline (PBS), the reacted slides were incubated in 1% normal goat serum for 30 min at room temperature to reduce nonspecific antibody binding and then incubated at 4 °C overnight with rabbit monoclonal antibody against GR (D6H2L, Cell Signaling Technology, Danvers, MA, USA, diluted 1/400), Sgk1 (Y238, Abcam, Cambridge, UK, diluted 1/200), or NDRG1 (EPR5593, Abcam, diluted 1/400). The reacted sections were then washed three times for 5 min each in PBS, incubated with biotinylated anti-rabbit immunoglobulin (Nichirei Biosciences, Inc., Tokyo, Japan), washed three times for 5 min each in PBS, and incubated with peroxidase-labeled streptavidin (Nichirei Biosciences, Inc.) for 30 min at room temperature. Immunoreactivity was visualized with 3,3 ′ -diaminobenzidine, and the slides were counterstained with Mayer's hematoxylin, dehydrated in graded alcohol, and cleared in xylene.