Alexa fluor anti mouse secondary

Cell proliferation was assessed by the 5-Bromo-2′-deoxyuridine (BrdU) assay which measured DNA synthesis by incorporation of the thymidine nucleotide analog BrdU (Thermo Fisher). Cos-7 cells (OE, EV, and WT) were seeded on coverslips in 6-well plates at a concentration of 1.5 × 10ˆ5. The cells were incubated with 10 μM BrdU solution for 2 h at 37 °C in the presence or absence of EGF. The cells were washed in PBS (3×) and fixed with 3% glyoxal for 10 min. The fixed cells were washed (3×) and permeabilized with 0.1% Triton X-100-PBS for 7 min. Following washing (6×), the DNA was hydrolyzed with 1 M HCl at RT for 30 min followed by blocking and immunostaining with an anti-BrdU monoclonal antibody (1:100; Thermo Fisher; MA3-071) for 2 h at RT. Finally, the cells were incubated with Alexa Fluor anti-mouse secondary for 30 min at RT (1:200; Thermo Fisher, A32728). Nuclei were stained with DAPI for 10 min at RT (Sigma, D9542). The cells were imaged on a Nikon Ti2 microscope using a 20× objective by collecting 10 fields of view per coverslip (n = 30 fields, across three independent experiments), and BrdU positive cells were manually counted.

Wings from male apterous-GAL4;UAS-disp flies were mounted and imaged using a Zeiss ICc3 camera and processed using Adobe Photoshop. Multiple male and female progeny from at least two independent crosses were analyzed. Representative wings were imaged. For salivary gland analysis, salivary glands were dissected from SGS-GAL4; UAS-HhGFP, UAS-V5dispHA third instar larvae using standard methods and immunostained as described (Carroll et al., 2012 (link)). Disp was detected using anti-HA (1:2000) and AlexaFluor anti-mouse secondary (1:10,000, Thermofisher). Multiple salivary glands from male and female larva were examined. A representative image is shown.