Alexa fluor 488 and alexa fluor 549 labeled secondary antibodies

Cells were fixed with 4% paraformaldehyde (Thermo Fisher Scientific) in PBS, permeabilized with 0.1% Triton X-100 (Thermo Fisher Scientific) in PBS, and then blocked in 3% BSA (Sigma-Aldrich). Subsequently, cells were incubated with specific primary antibodies overnight at 4 °C. The following primary antibodies at indicated dilutions were used: microcystin-LR (Creative Biolabs, 1:50), Vimentin (Cell Signaling Technology, 1:200), E-cadherin (Cell Signaling Technology, 1:200), Fibronectin (Abcam, 1:200), αSMA (Abcam, 1:200), GRP78 (Abcam, 1:200). Cells were stained with Alexa Fluor 488- and Alexa Fluor 549-labeled secondary antibodies (Thermo Fisher Scientific, 1:300) for 1 h at room temperature in the dark. Nuclear staining was performed with DAPI (Thermo Fisher Scientific, 1 μg/ml in PBS) for 10 minutes at room temperature. The images were observed on an Olympus confocal laser scanning microscope imaging system and a Zeiss fluorescent microscope.

Cells were washed twice in ice-cold PBS without calcium or magnesium, fixed in 4% paraformaldehyde (Thermo Fisher Scientific), and permeabilized with 0.1% Triton X-100 (Thermo Fisher Scientific). Cells were then incubated in 3% BSA (Sigma-Aldrich) to block cross-reactivity, and probed with specific primary antibodies for overnight at 4°C. The following primary antibodies were used at the indicated dilutions: MC-LR (Alexis Biochemicals, 1:200), vimentin (CST, 1:200), E-cadherin (CST, 1:200), fibronectin (Abcam, 1:200), and αSMA (Abcam, 1:200). The next day, cells were washed and stained with Alexa Fluor 488- and Alexa Fluor 549-labeled secondary antibodies (Thermo Fisher Scientific, 1:300) for 1 h at room temperature in the dark. Nuclear staining was performed with 4′,6-diamidino-2-phenylindole (DAPI; 1 μg/ml in PBS, Thermo Fisher Scientific) for 10 min at room temperature. Cells were washed, mounted onto glass slides, and visualized using an Olympus confocal laser scanning microscope imaging system and a Zeiss fluorescent microscope.