Figure 3-1
Validation of the antibody used for immunofluorescent staining of cultured hippocampal neurons. DIV 7 cultures were infected with lentivirus expressing either GFP (control) or GFP + shATF4, and fixed and stained on DIV 21. For ATF4 labeling, following fixing, cells were probed as described in Materials and Methods with ATF4 antibody (1:300; Cell Signaling Technology), rabbit monoclonal antibody D4B8 (catalog #11815, Thermo Fisher Scientific), and then a secondary anti-rabbit antibody, Alexa Fluor 568 (1:500; Thermo Fisher Scientific) after permeabilization. For β-III-tubulin, labeling was conducted with mouse monoclonal antibody TU-20 (1:300; catalog #NB-600-1018, Novus Biologicals), then a secondary anti-mouse antibody, Alexa Fluor 647 (1:500; Thermo Fisher Scientific). Top, Staining as imaged on an upright fluorescent microscope. Bottom, Staining as imaged by confocal microscopy (merged z-stack). Download Figure 3-1, TIF file.