Alexa fluor 594 goat anti mouse secondary antibody

The HeLa cells were cultured on sterile coverslips in a 12-well plate. The cells were incubated with the peptides (5 µM of R9 or Chk1-NP) for 2 h at 37°C in 5% CO₂ humidified air and washed 3 times with phosphate-buffered saline (PBS; Thermo Fisher Scientific Inc.). The cells were then fixed with methanol, stained with anti-Chk1 antibody (mouse monoclonal antibody against human Chk1, sc-8408; Santa Cruz Biotechnology, Inc.) and Alexa Fluor 594 goat anti-mouse secondary antibody (A-11005; Invitrogen™, Grand Island, NY, USA), washed with PBS, and re-stained with FITC-conjugated streptavidin (SA100-02) (both from Invitrogen) before staining with 4′,6-diamidino-2-phenylin-dole (DAPI). The nuclei were fluorescently labeled with DAPI (D9542; Sigma-Aldrich, Inc., St. Louis, MO, USA). The cells were then imaged using a Leica DMIRB microscope (Leica Microsystems Inc., Wetzlar, Germany) with a X20 objective. The fluorescent images were merged. HJURP was also detected by fluorescence microscopy as a nuclear resident protein and compared with localization of Chk1.

Using the standard protocol, immunofluorescence staining was conducted with primary antibodies targeting mouse anti-CD3 (1:250, Cat #MA5–12577; Invitrogen™, CA, USA), mouse anti-CD68 (1:200, Cat #MA5–13324; Invitrogen™), rabbit anti-Zonula occludens-1 (ZO-1) (1:200, Cat #61–7300; Invitrogen™) and rabbit anti-Claudin-1 (1:200, Cat #PA5–16833; Invitrogen™). After the samples had been incubated with the primary antibodies overnight at 4°C, they were washed with PBS and then probed with the Alexa Fluor™ 594 goat anti-mouse secondary antibody (1:500, Cat #A11005; Invitrogen™), Alexa Fluor™ 488 goat anti-rabbit secondary antibody (1:500, Cat #A27034; Invitrogen™), and Alexa Fluor™ 594 goat anti-rabbit secondary antibody (1:500, Cat #A11012; Invitrogen™) for 1 h at 37°C in the dark. The samples were counterstained with DAPI and observed by laser scanning confocal microscopy (Zeiss LSM880, Jena, Germany).