5.1 (Media Cybernetics, Silver Spring, MD). Data were stored in Adobe selleck products Photoshop, version 3.0, to enable uneven illumination and background color to be corrected. The number of cross sections of vWF and α-SMA-stained vessels and ED-1-stained macrophages was counted, and these numbers per square millimeter of the lesion were calculated, as described by Nap et al. (2004) . A semiquantitative evaluation of immunohistochemical staining for VEGF and Flk-1 was performed according to the method described by Donnez et al. (1998) . This method involves the analysis of the distribution and the intensity of staining within the endothelium and glandular epithelium or
stroma. The histologic scores (H) for VEGF and Flk-1 were calculated using the formula H = ΣPi, where i is the intensity ranging from 0 (negative cells) to 3 (deeply staining cells) and P is the percentage of staining cells for each given i, with P values of 1, 2, 3, Lazertinib purchase 4, and 5 indicating <15%, 15-50%, 50-85%, >85%, and 100% positive-staining cells, respectively. The staining result was expressed as mean ± standard Metabolism inhibitor deviations. Statistical Analyses All statistical calculations were carried out using the Stat-Xact-5 software program (CYTEL Software Corporation, Cambridge, MA). The differences between groups were calculated using nonparametric analyses (Mann-Whitney
U test). A P value of < 0.05 was established as statistically significant. Reverse transcription-polymerase chain reaction (RT-PCR) To investigate the expression of VEGF and Flk-1 and MMP-9 in eutopic endometrium and in endometriotic lesions, RT-PCR was performed. Total RNA was extracted from the tissues in TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. The purity and integrity of the RNA were checked by gel electrophoresis. One microgram of total RNA was subjected to reverse transcription with a commercially available kit (the cDNA First Chain Amplification System, GIBCO-BRL)
according to the manufacturer’s Molecular motor protocol. Amplification for VEGF cDNA was started with a 4-minute denaturation at 95°C followed by cycles of 30 seconds of denaturation at 94°C, 45 seconds of annealing at 61°C, and 45 seconds of extension at 72°C. The PCR profile for Flk-1 began with the 4-minute initial denaturation at 95°C, followed by cycles of 30 seconds of denaturation at 94°C, 45 seconds of annealing at 58°C, and 45 seconds of extension at 72°C. Amplification for MMP-9 cDNA was performed according to the following profile: initial denaturations at 94°C for 5 min, then 30 cycles at 94°C for 1 min 30 s, 63°C for 2 min and 72°C for 1 min. Transcripts were quantified after normalization with the endogenous control (GAPDH). Amplification for GAPDH cDNA was started with a 4-minute denaturation at 94°C followed by cycles of 30 seconds of denaturation at 95°C, 45 seconds of annealing at 63°C, and 45 seconds of extension at 72°C.