Nlrp2 is Required for Early Embryonic Development Figure 12. Effects of Nlrp2 overexpression on the expression of CDX2 and OCT3/4 and incidence of apoptosis in blastocysts. (A) Immunostaining of CDX2 (red) and OCT3/4 (green) in morulae and blastocysts developing from zygotes that had been microinjected with pIRES2 or pIRES2-Nlrp2. The original magnification was 6200. (B) TUNEL apoptosis assay of blastocysts (green). Each sample was counterstained with DAPI to visualize DNA (blue). The original magnification was 6200. (C) Number of apoptotic cells in each blastocyst. *p,0.05. doi:10.1371/journal.pone.0030344.g012 0.1% Tween 20 containing 5% dry milk following by an incubation overnight at 4uC with an anti-NLRP2 antibody (Sigma-Aldrich) at a 1:1000 dilution, After several washes in TBS/Tween 20, membranes were incubated for 2 h at RT with a horseradish peroxidase-linked secondary antibody goat anti-rabbit (Pierce, Rockford, IL, USA) at a 1:2000 dilution. The membrane was washed five times in TBS/Tween 20. Finally, the enhanced chemiluminescence (ECL) Advance Western Blotting detection system (Pierce) was used according to the manufacturer’s instructions. Detection of actin, with anti-b actin polyclonal rabbit antibody (1:1000, Santa Cruz Biotechnology Inc., sc-130656, Santa Cruz, CA, USA) and secondary anti-rabbit antibody (1:5000, Pierce) was used as a loading control. Immunofluorescence Oocytes and embryos were fixed in 4% paraformaldehyde in PBS for 45 min and permeabilized in PBS, 0.2% Triton X-100 for 10 min at RT. After a treatment with 3% BSA in PBS for 1 h, samples were incubated with the primary antibody diluted in PBS containing 1% BSA overnight at 4uC. Rabbit anti-NLRP2 antibodies (Sigma) were diluted 1:100, anti-CDX2 rabbit poly- clonal IgG (Santa Cruz, sc-134468) and anti-OCT3/4 mouse monoclonal antibody (Santa Cruz, sc-5279) was diluted 1:50, respectively. After washing in PBS containing 0.3% polyvinylpyr- rolidone (PBS/0.3% PVP), samples were incubated 1 h at RT with the secondary antibodies of Cy3-labeled goat anti-rabbit-IgG (Beyotime, A0516) for NLRP2, Alexa Fluor 555-labeled goat anti- PLoS ONE | www.plosone.org 12 January 2012 | Volume 7 | Issue 1 | e30344