Quantifying Autophagy Markers via Immunofluorescence

The levels of Microtubule-associated protein 1 light chain 3 (LC3)-II and lysosomal associated membrane protein (LAMP)-1 were visualized through confocal microscopy following immunofluorescent staining. The cells were exposed to primary antibodies targeting LC3-II (1:400, Medical and Biological Laboratory Co., Ltd., Woods Hole, MA, USA) and LAMP-1 (1:400, Santa Cruz Biotechnology, Dallas, TX, USA). After washing with PBS, the sections were incubated with a 1:500 dilution of Alexa fluor 488-conjugated anti-mouse IgG antibody (Cell signaling) or Alexa fluor 594-conjugated anti-rabbit IgG antibody (1:500 dilution; Cell signaling) for 1 h in the dark at room temperature. Subsequently, the nuclei were stained with 4′,6-diamidino-2-Phenylindole (DAPI) for 5 min, and then the samples were mounted. For the negative control, the primary antibody was omitted, and the sections were incubated with the corresponding secondary antibodies and the detection solutions. A confocal microscope (LSM 700; Zeiss, Jena, Germany) was used to capture fluorescence images Immunofluorescent staining evaluation involved the calculation of mean fluorescence intensity (MFI) using the ImageJ software (https://imagej.nih.gov/ij/download.html, accessed on 1 June 2022) in a blinded manner [6 (link),22 (link),23 (link)].