Fitc conjugated goat anti mouse igg

5 x 10³ MEFs were grown on glass coverslips in multiwell plates and infected with 10 moi of HSV-1 or SFV. At 18h post-infection, the cells were fixed in 4% paraformaldehyde and permeabilized in 0.1% Triton X-100 (Sigma). The cells were then incubated with mouse monoclonal anti-gD or mouse monoclonal anti-cytochrome c and rabbit polyclonal anti-caspase-3 antibodies followed by FITC-conjugated goat anti-mouse IgG, (Millipore) and PE-conjugated goat anti-rabbit IgG, F(ab’)₂ fragment (Santa Cruz Biotechnology) secondary antibodies (1:200) for 90 min. Nuclei were stained with Hoechst 33334 (5 mg/ml; Sigma). The samples were directly viewed under a Leica DMRE fluorescence microscope.

Primary antibodies against H3K9me2 (Abcam) and secondary antibodies [FITC-conjugated goat anti-mouse IgG (Millipore)] were used for immunostaining. MEFs attached to coverslips were washed with PBS–Tween (0.05% Tween-20 in PBS), fixed in 4% paraformaldehyde in PBS for 15 min and permeabilized with 0.2% Triton X-100 in PBS–Tween at room temperature (RT) for 15 min. The cells were further treated with 2 M HCl at RT for 30 min and subsequently washed in PBS–Tween, prior to incubation in blocking solution (2% BSA in PBS–Tween) for 60 min at RT. Cells were then incubated overnight at 4°C with the primary antibody diluted 1:200 in blocking buffer. The cells were washed three times for 5 min in PBS–Tween and incubated for 1 h at RT with the secondary antibody, diluted 1:400 in blocking buffer. DNA was stained with 5 mg/ml Hoechst 33342 for 2 min. The cells were washed three times for 5 min in PBS–Tween and mounted on to glass slides. Cell samples were examined using a confocal microscope (Leica TCS SP5). The experiment was repeated three times and normal serum without primary antibody was used as a negative control. Leica Microsystems software (LAS AF Lite Version: 1.8.1 build 1390) was used for quantitative analysis and the methylation status was calculated as the intensity of FITC staining divided by the intensity of nuclear staining with Hoechst 33342 [15 (link)].

Embryos were washed three times in phosphate-buffered saline/polyvinyl alcohol (PBS/PVA), fixed in 4% Paraformaldehyde (PFA) for 20 min. After washing extensively, embryos were permeabilized with 1% Triton-X-100 for 30 min at RT. Then, the unspecific binding sites in embryos were blocked by treating with blocking solution (PBS/PVA containing 0.1% Triton-X-100 + 1% BSA + 10% goat serum). Embryos were then incubated for overnight at 4°C with ß-CATENIN primary antibody (Anti-B-catenin antibody (E5); sc-7963, Santa Cruz). An appropriate negative control was carried out by omitting the primary antibody and using mouse IgG (S1 Fig). After washing extensively, embryos were incubated with FITC conjugated goat anti-mouse IgG (Millipore; AP124F) for 1 hour at 37ºC in the dark. The nucleus was labelled by 1 μg/ ml Hoechst 33342 for 15 min at RT. Embryos were finally rinsed in PBS/PVA and placed on a slide containing mounting solution, covered with a coverslip, and observed with a fluorescence microscope (Olympus BX51, Tokyo, Japan). The images were captured through a sensitive camera (Olympus DP71). At least 20 blastocysts (in total) in three replicates were used for differential staining. The fluorescence intensity was assessed by Image J software (National Institutes of Health, Bethesda, MD) and then was normalized by the area in each individual embryo.