Nlrp2 is Required for Early Embryonic Development Figure 11. Overexpression of Nlrp2 in zygotes. (A) Representative micrographs of blastocysts developing from zygotes that had been microinjected with pIRES2 or pIRES2-Nlrp2. Fluorescence images show the expression of GFP. The original magnification was 6100. (B) The blastocyst formation rate of zygotes microinjected with pIRES2 or pIRES2-Nlrp2. (C) The relative abundance of Nlrp2 transcripts after microinjection. (D) Immunoblots of mouse embryos at different stages after microinjection. doi:10.1371/journal.pone.0030344.g011 Immunohistochemistry Ovaries were collected from 3-week-old mice and fixed in 4% paraformaldehyde overnight, dehydrated through graded alcohol solutions and embedded in paraffin wax. Six-micrometer sections were deparaffinized in xylene, rehydrated through graded alcohol solutions and heated in 10 mM sodium citrate buffer (pH 6.0) with microwaves (15 min at medium power) and cooled to room temperature for antigen retrieval. Sections were then incubated with 10% nonimmune goat serum (Maixin-Bio, Kit-7770, Fuzhou, China) to block nonspecific sites followed by incubation with the NLRP2 polyclonal antibody (Sigma-Aldrich, SAB3500325) in blocking solution (1:200 dilution of rabbit anti-NLRP2 antibody; Beyotime, P0102, Jiangsu, China) overnight at 4uC. For enzymatic staining, sections were incubated for 10 min at RT with a biotinylated goat anti-rabbit secondary antibody (Maixin-Bio). Following three 5 min washes in PBS buffer with 0.1% Triton X- 100 (PBST), sections were incubated at RT for 10 min with streptavidin-linked alkaline phosphatase (Maixin-Bio) and then washed three times for 5 min each in PBST. 5-bromo-4-chloro-3- indolyl phosphate/nitro blue tetrazolium (BICP/NBT) substrate solution (Maixin-Bio) was applied to the sections for 10–30 min and the reaction was stopped by rinsing in PBS. Sections were then mounted with AEC Mounting Solution (Maixin-Bio, AEC- 0038). Negative controls consisted of identical reactions with the omission of the primary antibody. Immunoblotting To determine protein levels, fifty oocytes or embryos were loaded per lane. Total protein contents were separated by SDS- polyacrylamide gel (8%) and transferred to a PVDF membrane (Millipore, Bedford, MA) using semi-dry transfer. The membrane was blocked for 4 h at RT in Tris-buffered saline (TBS pH 7.4)/ PLoS ONE | www.plosone.org 11 January 2012 | Volume 7 | Issue 1 | e30344