Cells were plated on sterile glass coverslips and processed as in [6 (link)]. The following primary antibodies were used: mouse monoclonal anti-p62 (Becton Dickinson; dilution 1:200) and rabbit polyclonal anti-LC3 (Sigma-Aldrich; dilution 1:1000), mouse monoclonal anti-Vimentin (Abcam, dilution 1:100), and rabbit-polyclonal anti-Calreticulin (Thermo-Fisher, dilution 1:50). As secondary antibodies (dilution 1:1000), goat anti-mouse or anti-rabbit antibodies conjugated with AlexaFluor 488 or AlexaFluor 555 dye (Cell Signaling Technology Inc., Danvers, MA, USA) were used. Nuclear chromatin was stained with a fluorescent dye, 4,6-diamidino-2-phenylindole dihydrochloride (DAPI, Sigma-Aldrich), at 5 μg/mL. Images were acquired using a Nikon Ti2-E inverted microscope equipped with a DS-Qi2Mc camera and collected with a Nikon ×60 Plan Apocr λ (NA = 1.40) oil immersion objective, using FITC, TRITC, and DAPI detection filter sets.
For the LC3-p62 colocalization, images were processed using the freely available software ImageJ (version 1.53j), and a threshold-based analysis was performed to reveal the degree of overlap between channels. Colocalization was quantified as the fold change in autophagosomes loaded with cargo (yellow areas) normalized to the green channel area (total cargo signal).
For the LC3-p62 colocalization, images were processed using the freely available software ImageJ (version 1.53j), and a threshold-based analysis was performed to reveal the degree of overlap between channels. Colocalization was quantified as the fold change in autophagosomes loaded with cargo (yellow areas) normalized to the green channel area (total cargo signal).
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