Paraffin-embedded pathological specimens were obtained from the surgically resected tissues. All of these resection samples were treated with a standard fixation, dissection, and processing protocol. In addition, 300 pericarcinoma tissue samples were collected as a control group.
To examine the typical pathological changes, a large tissue microarray (TMA) was used. TMA blocks were constructed using a tissue arraying instrument (Beecher Instruments, Sun Prairie, WI, USA). Cylinders (1.5 mm in diameter) of representative areas of a tissue block were punched from the center of the tumor away from the areas of ulceration and necrosis and re-embedded in a defined position within a recipient paraffin block. The TMA blocks were then cut into 4-mm sections and processed for immunohistochemistry (IHC).
After being washed with phosphate-buffered saline, the specimens were incubated with the primary antibody using ER antibody (dilution 1:50), PR antibody (dilution 1:50), HER2 antibody (dilution 1:50), and P-p38 MAPK antibody (dilution 1:40; Cell Signaling Technology Inc. Boston, USA). Immunostaining was conducted using the Envision System with diaminobenzidine (Dako, Glostrup, Denmark). A negative control was obtained by replacing the primary antibody with normal murine or rabbit immunoglobulin G with the same dilution.
To examine the typical pathological changes, a large tissue microarray (TMA) was used. TMA blocks were constructed using a tissue arraying instrument (Beecher Instruments, Sun Prairie, WI, USA). Cylinders (1.5 mm in diameter) of representative areas of a tissue block were punched from the center of the tumor away from the areas of ulceration and necrosis and re-embedded in a defined position within a recipient paraffin block. The TMA blocks were then cut into 4-mm sections and processed for immunohistochemistry (IHC).
After being washed with phosphate-buffered saline, the specimens were incubated with the primary antibody using ER antibody (dilution 1:50), PR antibody (dilution 1:50), HER2 antibody (dilution 1:50), and P-p38 MAPK antibody (dilution 1:40; Cell Signaling Technology Inc. Boston, USA). Immunostaining was conducted using the Envision System with diaminobenzidine (Dako, Glostrup, Denmark). A negative control was obtained by replacing the primary antibody with normal murine or rabbit immunoglobulin G with the same dilution.