Alexa fluor 488 f ab 2 fragment of goat anti rabbit igg antibody

AGS-BDneo and HA cells grew on cover slips were treated with drugs for specific duration depending on experimental needs. Cells were fixed with acetone for 10 mins at room temperature. The fixed cells were then stained with anti-Zta, cleaved caspase-3, or LC3B rabbit polyclonal antibody (1:200; Cell Signaling Technology, Beverly, MA) overnight at 4 °C. Expression of the proteins was visualized with Alexa Fluor 488 F(ab′)2 fragment of goat anti-rabbit IgG antibody (1:500; Invitrogen) under fluorescence microscopy or Carl Zeiss LSM 710 confocal microscope (Carl Zeiss, Oberkochen, Germany). Nuclei of cells were stained with 4′,6-diamidino-2-phenylindole (DAPI) (Roche, Mannheim, Germany).

HONE-1 and HA cells grew on cover slips were treated with drugs for specific duration depending on experimental needs. Cells were first washed with PBS once and were fixed with ice-cold acetone for 10 min at room temperature. The fixed cells were then washed with PBS twice and were blocked with 5% normal goat serum (PCN5000, Gibco, Gaithersburg, MD, USA) in 1× TBST (50 mM Tris, 150 mM NaCl, 0.1% Triton-X, pH 7.4) for 30 min at room temperature. After that, the cells were stained with anti-Zta monoclonal antibody (1:50), LAMP-1, cleaved caspase-3, or LC3B rabbit polyclonal antibody (1:200; Cell Signaling Technology, Beverly, MA, USA) in 5% normal goat serum, 1× TBST overnight at 4 °C. Expression of the proteins was visualized with Alexa Fluor 594 F(ab′)2 fragment of goat anti-mouse IgG antibody or Alexa Fluor 488 F(ab′)2 fragment of goat anti-rabbit IgG antibody (1:500; Invitrogen) under fluorescence microscopy. Nuclei of cells were stained with 4′,6-diamidino-2-phenylindole (DAPI) (Roche, Mannheim, Germany).

GC cells grown on cover slips coated with 0.1% gelatin were treated with drugs for 24 hr. Cells were fixed with acetone for 10 minutes at room temperature. The fixed cells were then stained with cleaved poly (ADP-ribose) polymerase (PARP) or p62/SQSTM1 rabbit polyclonal antibody (1:200; Cell Signaling Technology, Beverly, MA) overnight at 4°C. Expression of proteins was visualized with Alexa Fluor 488 F(ab')2 fragment of goat anti-rabbit IgG antibody (1:500; Invitrogen) under fluorescent microscope or Carl Zeiss LSM 710 confocal microscope (Carl Zeiss, Germany). Nuclei of cells were stained with 4′,6-diamidino-2-phenylindole (DAPI) (Roche, Mannheim, Germany).