For tissue immunofluorescence, slides were deparaffinized and hydrated. Tris-EDTA pH 9.0 was utilized for antigen retrieval. Intrinsic peroxidase activity was inactivated, and serum blocking was performed at room temperature. CD31, E-cadherin, Vimentin and RBM39 antibodies were added to the tissues and incubated for 13 h at 4 °C. Antibody concentrations were all diluted 1:200. Next, the slides were washed and Alexa Fluor-labeled secondary antibodies (Abcam) were added to the slides for incubation. Finally, the tissues were stained with DAPI solution for nuclei and photographed with a confocal microscope (Leica).
For cellular immunofluorescence, cells were first fixed with 4% paraformaldehyde. Then, serum blocking was performed at room temperature. E-cadherin, Vimentin and RBM39 antibodies were added to the cells and incubated for 13 h at 4 °C. Antibody concentrations were all diluted 1:200. Next, the cells were washed and Alexa Fluor-labeled secondary antibodies (Abcam) were added to the cells. Finally, the cells were stained with DAPI solution for nuclei and photographed using a confocal microscope (Leica).
For cellular immunofluorescence, cells were first fixed with 4% paraformaldehyde. Then, serum blocking was performed at room temperature. E-cadherin, Vimentin and RBM39 antibodies were added to the cells and incubated for 13 h at 4 °C. Antibody concentrations were all diluted 1:200. Next, the cells were washed and Alexa Fluor-labeled secondary antibodies (Abcam) were added to the cells. Finally, the cells were stained with DAPI solution for nuclei and photographed using a confocal microscope (Leica).