Number of cells and differential labeling. To determine to what extents the numbers of cells and ICM:TE ratio were affected by treatment with neutralizing antibodies, ICM and TE cells were differentially labeled using PI and bisbenzimide (Hoechst 33258) by using a modification of a previously published protocol. Briefly, blastocysts with intact zona pellucida were incubated in 500 ц1 HEPES-buffered HTF (Specialty Media) containing 1% Triton X100 and 100 |ig/ml PI for 10 s, during which time changes in the color and size of TE cells could be seen microscopically. Blastocysts were transferred immediately into 500 ц1 100% ice-cold ethanol containing 25 |ig/ml bisbenzimide and incubated overnight at 4 C. Fixed and stained blastocysts were mounted on a glass slide in 5 ц1 glycerol and gently flattened with a coverslip, and fluorescence was visualized under a fluorescence microscope. To analyze the total number of cells, cells were visualized using a filter for DAPI (330 to 380 nm), whereas differentially labeled ICM and TE cells were visualized using different filters (460 nm for blue and red fluorescence, and 560 nm for red only).
ICM:TE ratio. The ratio of ICM:TE cells is an indication of differences in the pattern of the cell division and rate of cell death between the 2 cell lineages in early preimplantation embryos (blastocysts). Compared with that after control antibody treatments, the ICM:TE ratios (mean + SE) of in vivo-derived embryos did not differ (P = 0.097) after treatment with individual neutralizing antibodies but were significantly (P < 0.01) different after combined antibody treatment (1:3.16 + 0.03; Table 4, Figure 1 F). In contrast, significantly (P < 0.05) different relative results were observed after treatment of in vitro-fertilized embryos with neutralizing antibodies to EGF (1:2.94 + 0.02), TGFa (1:2.96 + 0.02), and EGFR (1:2.95 + 0.02) and combined treatment with antibodies to both EGF and TGFa (1:3.24 + 0.02). Further, after combined antibody treatment, the mean ICM:TE ratios of fertilized embryos were not significantly different from those of cloned embryos (1:3.21 + 0.03; Table 4, Figure 1 C, F, and I). Welcome to the pharmacy that will make your symptoms go away and still leave you with plenty of money to spend. Сanadian neighbor pharmacy takes pride in offering only highest quality medicine at best prices, because we work directly with leading manufacturers and can guarantee the quality of every pill you take, every time you need to.
Table 1. Rate and percentage (mean ± SE) of development of 1-cell stage to blastocyst stage of in vivo-derived, in vitro-derived, and ICNI-derived (cloned) embryos after treatment with neutralizing antibodies to EGF, TGFa, and EGF-R; combined treatment with EGF and TGFa; and negative (no antibody) and positive (anti-KLH) control treatments
Symbols indicate statistically significant (*, P < 0.05; +, P < 0.01) differences from control treatments within an embryo group