Alexa 594 labeled anti rat secondary antibody

After treatments followed by fixation, the cells were incubated with rabbit anti-Rab7a antibody (1:100; Abcam Biotechnology, Cambridge, United Kingdom) and rat anti-Lamp-1 antibody (1:100; Santa Cruz Biotechnology, Dallas, TX, USA) overnight at 4 °C. After washing the slides, Alexa 488-labeled anti-rabbit secondary antibody (1:200; Life Technologies, CA, USA) and Alexa 594-labeled anti-rat secondary antibody (1:200; Life Technologies, CA, USA) were added to the cell slides and incubated for 1 h at room temperature. Slides were then washed, stained with DAPI, and mounted. A Nikon fluorescence microscope in the structured illumination microscopy (SIM) mode was used to obtain images. Image Pro Plus 6.0 software (Media Cybernetics, Bethesda, MD, USA) was employed to analyze colocalization, expressed as the Pearson correlation coefficient (Li, Huang, Li, Ritter, & Li, 2021 (link)).

After treatments followed by fixation, the cells were incubated with rabbit anti-LC3 antibody (1:100; Cell Signaling Technology, Danvers, MA, USA) and rat anti-Lamp-1 antibody (1:100; Santa Cruz Biotechnology, Dallas, TX, USA) overnight at 4 °C. After slides being washed, Alexa 488-labeled anti-rabbit secondary antibody (1:200; Life Technologies, CA, USA) and Alexa 594-labeled anti-rat secondary antibody (1:200; Life Technologies, CA, USA) were added to the cell slides and incubated for 1 h at room temperature. Slides were then washed, stained with DAPI, and mounted. A Nikon fluorescence microscope in the structured illumination microscopy (SIM) mode was used to obtain images. Image Pro Plus 6.0 software (Media Cybernetics, Bethesda, MD, USA) was employed to analyze colocalization, expressed as the Pearson correlation coefficient [28 (link),39 (link)].