Fig 2: Relative gene expression and localization of TLR4, IDO1 and AIF1 in the canine uterus. (A–C) Relative gene expression as determined by semi-quantitative real time (TaqMan) PCR (mean ± SEM). One-way ANOVA was applied to test the variation among the investigated groups, revealing: P = 0.0033 for TLR4, P = 0.03 for IDO1, and P = 0.04 for AIF1. In the case of P < 0.05, this was followed by Tukey-Kramer multiple comparisons post-test. Bars with asterisks differ at: *P < 0.05, **P < 0.01. (D–M) Immunohistochemical localization of members of the TNF-system, IDO1, and AIF1 in the canine uterus at selected stages of early pregnancy. Signals of TNFa were present in macrophages during the pre-implantation (D) and implantation (E) periods. Similarly, both TNFR1 (F) and TNFR2 (G,H) were present in macrophages. In addition, weaker signals for both receptors were also observed in epithelial cells of uterine glands during pre-implantation (F,G) and in endothelial cells (F,H). Positive signals of IDO1 were detected in macrophages between superficial glands during pre-implantation (I) and were also detected in endothelial cells at the time of implantation (J). At post implantation, weaker signals were observed in macrophages localized in deep endometrium layers, as well as in endothelial cells (K). AIF1 positive signals were identified in macrophages close to superficial uterine glands in pre-implantation (L) and implantation (M) stages, while at post-implantation they were localized in the deep layer of the endometrium (N). Some weak signals were also observed in uterine glands at the pre-implantation period (L), while apparently stronger signals were observed in the same glands at implantation (M) and post-implantation (N). Weak positive signals were also detected in endothelial cells (M, N) (solid arrow = macrophages; closed arrowhead = uterine gland; open arrowhead = blood vessel). No staining is observed in the isotype controls [inset in (D,F,G,I,L)].