Serial sections were cut at 8 ��m thickness on a cryostat and mounted on poly-L-lysine-treated slides. They were stained with selleckchem Ixazomib hematoxylin-eosin (HE) and observed under microscopy (BX50; Olympus, Japan). The histological diagnosis of endometriosis was based on the morphological identification of endometriotic glands and stroma. The weight of endometriotic lesions was measured. Lipid mediator analyses LC-MS/MS-based mediator lipidomics was performed as described previously [26]. Briefly, samples were extracted by solid-phase extraction using Sep-Pak C18 cartridges (Waters, Milford, MA, USA) with deuterium-labeled internal standards (PGE2-d4, LTB4-d4, 15-HETE-d8, arachidonic acid-d8). LC-MS/MS-based lipidomic analyses are performed on Acquity UPLC BEH C18 column (1.0 mm��150 mm��1.

7 ��m) using Acquity UltraPerformance LC system (Waters Co.) coupled to an electrospray (ESI) triple quadrupole mass spectrometer (QTRAP5500; AB SCIEX). The MS/MS analyses were performed in negative ion mode, and the eicosanoids and docosanoids were identified and quantified by multiple reaction monitoring. Calibration curves between 1 and 1000 pg and the LC retention times for each compounds were constructed with synthetic standards. cDNA Microarray We collected cystic lesions 2 weeks after injection of minced endometrium, and total RNA was extracted from their lesions. they were incubated in RNA later. For the cDNA microarray analysis, 0.5 ��g of pooled total RNA was amplified and labeled using an Amino Allyl MessageAmpTM II aRNA Amplification kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer��s instructions.

Each sample of aRNA labeled with Cy3 and reference aRNA labeled with Cy5 was cohybridized with GeneTM Mouse Oligo chip 24 k (Toray Industries Inc., Tokyo, Japan) at 37��C for 16 h. After hybridization, each DNA chip was washed and dried. Hybridization signals derived from Cy3 and Cy5 were scanned using Scan Array Express (PerkinElmer, Waltham, MA, USA). The scanned image was analyzed using GenePix Pro (MDS Analytical Technologies, Sunnyvale, CA, USA). All analyzed Brefeldin_A data were scaled by global normalization. RT-PCR for peritoneal macrophages After washing the peritoneal cavity of each mouse with 5 ml PBS, peritoneal washes were filtered through a 30 ��m strainer and centrifuged for 5 min at 200 g. From recovered cellular components, CD11b-positive macrophages were isolated using an isolation kit and incubated in RNA later. We isolated RNA from these macrophages after homogenizing. Two ��g of total RNA was extracted using an RNeasy Mini Kit (Qiagen, Hilden, Germany), followed by reverse transcription.