Alexa fluor plus 555 goat anti rabbit igg

Cells were cultured in chamber slides overnight. After washing with PBS, cells were fixed and permeabilized with 4% PFA in PBS containing 0.1% Triton X-100 for 20 min at room temperature. Cells were then blocked for non-specific binding with 1% BSA in PBS at room temperature for 1 h. After extensive washing in PBS, cells were incubated with the E-cadherin antibody (1: 200) and the Vimentin antibody (1: 300) for 3 h at room temperature, followed by incubation with Alexa Fluor Plus 555 goat anti-rabbit IgG (1: 1000, Invitrogen, A32732) and Alexa Fluor Plus 488 goat anti-mouse IgG (1: 1000, Invitrogen, A32723) for 45 min at room temperature. Cover slips were mounted on slides using anti-fade mounting medium with DAPI. Immunofluorescence images were acquired on an Olympus FV3000 fluorescence microscope.

Details regarding immunofluorescence experiments were performed the same as those described in previously studies [21 (link),32 (link)]. A total of 24 h after transfection, the cells were washed by PBS buffer three times for 5 min each time, fixed by 4% paraformaldehyde for 30 min and washed three times again by PBS buffer. Cells were treated by 0.4% Triton X-100 (Sangon Biotech, Shanghai, China) for 15 min and blocked by 10% FBS for 1 h. Immunostaining was performed by rabbit monoclonal anti-p62 antibody (1:200, Proteintech, Wuhan, China) in PBS containing 10% FBS and incubated at 4 °C overnight, followed by staining with Alexa Fluor Plus 555 goat anti-rabbit IgG (1:500, Invitrogen, Waltham, MA, USA) for 2 h at room temperature. The nuclei were stained with DAPI (#36308ES20, Yeasen, Shanghai, China). The exogenously expressed recombinant proteins were detected by the tagged GFP. All immunostaining images and the distribution of these proteins in the HeLa cells were visualized and captured by a Leica DMi8 (Leica, Wetzler, Germany) confocal laser microscope. The percentage of cells with aggregates (cells with GFP highlight aggregation) was analyzed from at least 200 positively transfected cells in 10 randomly selected viewing fields using ImageJ (Collins 2007) and presented as average ± SD.