Ms 348 p

The expression of additional epithelial markers such as transmembrane mucin 1 (MUC1), also known as epithelial membrane antigen (EMA), and cytokeratin cocktail (AE1/AE3) were detected by immunohistochemistry methods. Cells were xed with 4% paraformaldehyde in PBS 1X, treated with 0.1% Triton x-100 in PBS for 15 min and blocked with 2% BSA in PBS at RT for 2 hours. Then, primary anti-EMA antibodies (MS-348-P, Thermo Scienti c, USA) and anti-AE1/AE3 (MA5-13156, Thermo Scienti c, USA) were incubated with cells at 4 o C for 16 hours at 1:100 dilution. Finally, the slides were washed in PBS and incubated with an HRP conjugated goat anti-mouse IgG secondary antibody at RT for 45 min. Immunocytochemical staining was performed using an avidin-biotin complex peroxidase standard staining kit. HeLa and AGS cell lines (ATCC CRL-1739) were used as positive controls for AE1/AE3 and MUC1 markers, respectively, whereas hematopoietic U-937 cell lines (ATCC ® CRL-1593.2 ™ ) were used as a negative control. Imaging was made using a Nikon Eclipse 2000 microscope (Nikon, Tokyo, Japan). All experiments were performed in triplicate.

The expression of additional epithelial markers such as transmembrane mucin 1 (MUC1), also known as epithelial membrane antigen (EMA), and cytokeratin cocktail (AE1/AE3) were detected by immunohistochemistry methods. Cells were xed with 4% paraformaldehyde in PBS 1X, treated with 0.1% Triton x-100 in PBS for 15 min and blocked with 2% BSA in PBS at RT for 2 hours. Then, primary anti-EMA antibodies (MS-348-P, Thermo Scienti c, USA) and anti-AE1/AE3 (MA5-13156, Thermo Scienti c, USA) were incubated with cells at 4 o C for 16 hours at 1:100 dilution. Finally, the slides were washed in PBS and incubated with an HRP conjugated goat anti-mouse IgG secondary antibody at RT for 45 min. Immunocytochemical staining was performed using an avidin-biotin complex peroxidase standard staining kit. HeLa and AGS cell lines (ATCC CRL-1739) were used as positive controls for AE1/AE3 and MUC1 markers, respectively, whereas hematopoietic U-937 cell lines (ATCC ® CRL-1593.2 ™ ) were used as a negative control. Imaging was made using a Nikon Eclipse 2000 microscope (Nikon, Tokyo, Japan). All experiments were performed in triplicate.