Goat anti ncf1 antibody

Cells were fixed with 4% paraformaldehyde (PFA) for 15 minutes and treated for 10 minutes with PBS containing Triton X-100 (0.25%). The samples were then incubated with mouse IgG–blocking reagent (BioLegend), followed by the addition of goat anti-NCF1 antibody (1:50, Abcam) and rabbit anti-EEA1 antibody (1: 100, Abcam) for 1 hour and then washing. AF488 donkey anti–goat IgG antibodies (1:100) were then stained for 30 minutes and washed. Finally, AF647 goat anti–rabbit IgG antibodies (1:100) were incubated for 30 minutes at room temperature. To detect the colocalization of NCF1 and LAMP1, AF647 rat-mouse antibody (1:100, BioLegend) and a goat anti-NCF1 antibody (1:50, Abcam) were incubated for 1 hour and washed. Then, AF488 donkey anti–goat IgG antibody (1:100) was stained for 30 minutes at room temperature. After that, cells were incubated with DAPI (1:5000, BD Biosciences) for 5 minutes. Cells were then detached onto glass dishes and viewed and photographed on a confocal fluorescence microscope (THUNDER Imager Tissue and Leica TCS SP8, Leica Microsystems). All immunofluorescence was assessed in a blinded manner by 2 observers throughout the analysis process. The colocalization of NCF1 and EEA1 and of NCF1 and LMAP1 was calculated with ImageJ software (NIH).