Gnificance of PKCa protein overexpression in gastric carcinoma was also investigated.Quantitative Real-Time PCR TestAt first quantitative real-time PCR test was applied to test and compare the mRNA expression of PKCa in tumorous and nontumorous tissues of gastric carcinoma in a small scale. Ten tumor and non-tumor pairs of gastric tissues were randomly selected from the Tumor and Serum Bank of Chi-Mei Medical Center (Tainan, Taiwan). All samples were collected from the specimens via 1655472 radical gastrectomy. The non-tumor part was taken from the grossly normal gastric mucosa away from the tumor. All tissues were frozen in liquid nitrogen within 20 min and kept at ?0uC until use. The procedure of quantitative real-time PCR test was performed according to previous study [15].Immunohistochemical StudySections of 5 mm thickness were taken from formalin-fixed paraffin-embedded blocks. The procedure of immunohistochemical study was performed according to previous study [15]. Deparaffinized sections were incubated in pH 6.0 citrate Hypericin buffer for 40 min at 95uC on a hotplate to retrieve the antigens. Endogenous peroxidase was blocked by 3 hydrogen peroxide for 5 min. The sections were subsequently incubated with antibody against PKCa (Santa Cruz Biotechnology Inc., Santa Cruz, CA, SC-8393) for 30 min at room temperature at a MedChemExpress AN-3199 dilution of 1:100 using DAKO primary antibody diluent. To detect immunoreactivity, the avidinbiotin-complex method was applied according to the manufacturer’s instructions. A sensitive Dako EnVision kit (Dako North America Inc., Carpinteria, CA) was used as the detection system. After incubation with secondary antibody (DAKO EnVision) for 30 min at room temperature, followed by diaminobenzidine for 8 min, sections were counterstained with Mayer’s hematoxylin. Normal human distal renal tubules were used as a positive control. The negative control was made by omitting the primary antibody and incubation with PBS. The PKCa immunoreactivity was evaluated independently by two pathologists (CL Fang and SE Lin). As in previous studies [16,17], the results were scored semiquantitatively in four categories: 0 = absent, 1 = weak, 2 = moderate, and 3 = strong immunoreactivity. The positive staining of nerve bundles in the same slide was used as the positive internal control and was allocated score 2. The negative control provided a reference of score 0. Score 1 was defined as positive staining that was weak compared with internal control; score 3 was allocated to positive staining stronger than that of internal control. Finally each case was assigned to one of two groups: either PKCa overexpression with score 2 or 3, or non-overexpression with score 0 or 1.Materials and MethodsWe collected 215 consecutive cases of gastric carcinoma from the medical files of both Wan-Fang Hospital and Taipei Medical University Hospital in Taiwan. All patients included in our study group were treated between 1997 and 2011, and had received surgical resection with radical total or subtotal gastrectomy and lymph node dissection. All pathological reports and hematoxylin eosin sections were available and reviewed to determine pathological parameters including tumor size, location, histologic type, differentiation, depth of invasion, angiolymphatic invasion, nodal status, local recurrence status, distant metastasis, and pathologic staging. The pathologic staging was based on the 7th edition of the TNM staging system of AJCC. For each case, one or more representat.Gnificance of PKCa protein overexpression in gastric carcinoma was also investigated.Quantitative Real-Time PCR TestAt first quantitative real-time PCR test was applied to test and compare the mRNA expression of PKCa in tumorous and nontumorous tissues of gastric carcinoma in a small scale. Ten tumor and non-tumor pairs of gastric tissues were randomly selected from the Tumor and Serum Bank of Chi-Mei Medical Center (Tainan, Taiwan). All samples were collected from the specimens via 1655472 radical gastrectomy. The non-tumor part was taken from the grossly normal gastric mucosa away from the tumor. All tissues were frozen in liquid nitrogen within 20 min and kept at ?0uC until use. The procedure of quantitative real-time PCR test was performed according to previous study [15].Immunohistochemical StudySections of 5 mm thickness were taken from formalin-fixed paraffin-embedded blocks. The procedure of immunohistochemical study was performed according to previous study [15]. Deparaffinized sections were incubated in pH 6.0 citrate buffer for 40 min at 95uC on a hotplate to retrieve the antigens. Endogenous peroxidase was blocked by 3 hydrogen peroxide for 5 min. The sections were subsequently incubated with antibody against PKCa (Santa Cruz Biotechnology Inc., Santa Cruz, CA, SC-8393) for 30 min at room temperature at a dilution of 1:100 using DAKO primary antibody diluent. To detect immunoreactivity, the avidinbiotin-complex method was applied according to the manufacturer’s instructions. A sensitive Dako EnVision kit (Dako North America Inc., Carpinteria, CA) was used as the detection system. After incubation with secondary antibody (DAKO EnVision) for 30 min at room temperature, followed by diaminobenzidine for 8 min, sections were counterstained with Mayer’s hematoxylin. Normal human distal renal tubules were used as a positive control. The negative control was made by omitting the primary antibody and incubation with PBS. The PKCa immunoreactivity was evaluated independently by two pathologists (CL Fang and SE Lin). As in previous studies [16,17], the results were scored semiquantitatively in four categories: 0 = absent, 1 = weak, 2 = moderate, and 3 = strong immunoreactivity. The positive staining of nerve bundles in the same slide was used as the positive internal control and was allocated score 2. The negative control provided a reference of score 0. Score 1 was defined as positive staining that was weak compared with internal control; score 3 was allocated to positive staining stronger than that of internal control. Finally each case was assigned to one of two groups: either PKCa overexpression with score 2 or 3, or non-overexpression with score 0 or 1.Materials and MethodsWe collected 215 consecutive cases of gastric carcinoma from the medical files of both Wan-Fang Hospital and Taipei Medical University Hospital in Taiwan. All patients included in our study group were treated between 1997 and 2011, and had received surgical resection with radical total or subtotal gastrectomy and lymph node dissection. All pathological reports and hematoxylin eosin sections were available and reviewed to determine pathological parameters including tumor size, location, histologic type, differentiation, depth of invasion, angiolymphatic invasion, nodal status, local recurrence status, distant metastasis, and pathologic staging. The pathologic staging was based on the 7th edition of the TNM staging system of AJCC. For each case, one or more representat.
