49 6 diamidino 2 phenylindole dapi

Lung cancer cell line A549 was grown on coverslips to 70% confluence, then all cells were fixed with 4% paraformaldehyde for 10 minutes and permeabilized with 0.5% TritonX-100 for 10 minutes after 24 hours. Blocking was performed with 3% Albumin Bovine V (A8020, Solarbio, Beijing, China) for 1 hour at the room temperature (RT). After washing with PBS, cells were incubated with antibody against NQO1 (1:200, Cell Signaling Technology, Boston, USA) for 2 hours at 37°C, and followed the incubation by Alexa Fluor®488 goat anti-rabbit IgG (H + C) (A11008, Invitrogen, USA) respectively, for 1 hour at RT. After washing with PBS, cells were counterstained with 49-6-diamidino-2-phenylindole (DAPI) (C1006, Beyotime, Shanghai, China) and the coverslips were mounted with Antifade Mounting Medium (P0126, Beyotime, Shanghai, China). Finally, the immunofluorescence signals were visualized and recorded by Leica SP5II confocal microscope.

The CRC cell line, SW-620, was grown on coverslips to 70% confluence, and then fixed in 4% paraformaldehyde for 10 min and permeabilized with 0.5% TritonX-100 for 10 min after 24 h. Blocking was performed with 3% bovine serum albumin fraction V (Solarbio, Beijing, China) for 1 h at room temperature. After washing with PBS, cells were incubated with mouse anti-human DEK (1∶50, BD Biosciences Pharmingen, San Diego, CA, USA) at 4°C overnight, followed by incubation with Alexa Fluor 568 goat anti-mouse IgG (H+L) (A11004, 1∶1000, Life Technologies, Carlsbad, CA, USA) for 1 h at room temperature. After washing with PBS, cells were counterstained with 49-6-diamidino-2-phenylindole (DAPI) (Beyotime, Shanghai, China) and the coverslips were mounted with Antifade Mounting Medium (Beyotime, Shanghai, China). Finally, immunofluorescence signals were visualized and recorded using Leica SP5II confocal microscope [21] (link).

IF staining was used to detect the sub-cellular localization of NQO1 protein in breast cancer cells. All steps were performed at RT. Breast cancer cells were grown on coverslips to 70–80% confluence, then fixed with 4% paraformaldehyde for 10 min and permeabilized with 0.5% TritonX-100 for 10 min after 24 h. After blocking with 3% Albumin Bovine V (A8020, Solarbio, Beijing, China) for 1 h, the slides were quickly and gently washed with PBS. The cells were then incubated with the E-cadherin, Vimentin antibody at 4 °C overnight, and followed by incubation with Alexa Fluor® 568 goat anti-mouse IgG (H + L) (A11004, 1:1000, Invitrogen, Carlsbad, CA, USA) for 1 h. After washing with PBS, cells were counterstained with 49–6-diamidino-2-phenylindole (DAPI) (C1006, Beyotime, Shanghai, China) and the coverslips were mounted with Antifade Mounting Medium (P0126, Beyotime)^{27 (link)}. Finally, the IF signals were visualized under a Leica SP5II CLSM microscope (Heidelberg, Germany) with filters for the corresponding fluorescent stains.