Goat anti mouse igg conjugated to fitc

At 144 h after infection, the infected DF-1 cells were washed twice with PBS and fixed with anhydrous ethanol for 20 min. The fixed cells were then incubated with mouse anti-P27 monoclonal antibody (prepared in our lab) at 37°C for 60 min. After washing three times with PBST (0.01 M PBS, pH 7.2, 0.05% Tween 20), the cells were incubated with goat anti-mouse IgG conjugated to FITC (Sigma, USA) at 37°C for another 60 min. Finally, the cells were observed under a Carl Zeiss Vision microscope (ZEISS Axio Observer D1) after three washes with PBST.

Cells were fixed in absolute methanol for 10 min at -20°C, washed in PBS and treated with 2 N HCl for 1 h at 37°C. The material was then washed twice in borate buffer (100 mM boric acid, 75 mM NaCl and 25 mM sodium tetraborate, pH 8.5) and blocked with 1% BSA in PBS for 1 h. Next, the cells were incubated with mouse anti-5-methylcytosine primary antibody (Sigma^®, 1:100 diluted in 1% BSA) for 1 h at room temperature in the dark, followed by treatment with goat anti-mouse IgG conjugated to FITC (Sigma, 1:50 diluted in 1% BSA) for 1 h in the dark.
Image capture was performed using an Olympus BX60F5 microscope, QCapture and Image Pro-Plus software, and the same exposure times. ImageJ (NIH, Bethesda, USA) software was used for image analysis.