For endometrial dating, 4 um sections stained with hematoxylin and eosin and periodic acid Schiff stain were evaluated. All endometrial biopsies were evaluated by the same expert pathologist according to the histopathological criteria of Noyes et al. The endometrium with endometrial maturity of cycle day 25 citation or less at the Inhibitors,Modulators,Libraries time of menstrua tion was considered out of phase. Endometrial tissues were fixed in 10% buffered formaldehyde. Apoptosis detection system For apoptosis quantification, sections were processed for in situ immunohistochemical localization of nuclei exhi biting DNA fragmentation, by the TUNEL technique, using the apoptosis detection kit Apoptag Plus. Sections were treated according to the manufacturers instructions as previously described.

Briefly, sections were deparaffinized and rehydrated with xylene and ethanol, and permeabilized with 20 ug ml Inhibitors,Modulators,Libraries Proteinase K. Endo genous peroxidase was inactivated by coating the samples with 3% H2O2. Sections were rinsed with PBS, and then immersed 60 min in TdT buffer at 37 C. Afterwards, they were incubated 30 min with the anti digoxygenin peroxidase conjugate, followed by the peroxidase sub strate diaminobenzidine. Finally, sections were counterstained with hematoxylin. Sections of female rodent mammary gland obtained 3 5 days after weaning of pups, were used as a positive control. As a negative control, a number of tissue samples were subjected to treatment without TdT. The percentage of apoptotic cells was determined by counting labeled cells at 400X magnification in 10 randomly selected and homogeneous fields.

Inhibitors,Modulators,Libraries Apoptotic cells were also identified by their characteris tic morphological features in hematoxylin eosin stained endometrial sections cell shrinkage and chromatin margination or chromatin condensation with Inhibitors,Modulators,Libraries formation of apoptotic bodies. The TUNEL method is characterized by higher sensi tivity than most other histochemical approaches and is considered to be the gold standard to detect apoptosis in situ. However, in order to complete the apopto sis evaluation we also included the assessment of the cleaved caspase 3, the most important effector enzyme in apoptosis pathway. Immunohistochemistry Serial sections of the endometrium were subjected to standard immunohistochemistry as previously described. Briefly, sections were deparaffinized in xylene and rehydrated through graded alcohols followed by micro waving Inhibitors,Modulators,Libraries in 0.

01 M sodium citrate buffer for antigen retrieval. Endogenous peroxidase was blocked by treat ment with 3% hydrogen peroxide for 10 min at room temperature, after which, non specific binding was blocked incubating with 4% bovine serum albumin in phosphate buffer for 60 min. Tissue sections were incu bated overnight with the then primary antibody rabbit anti human PCNA polyclonal. or rabbit anti human cleaved caspase 3 at 4 C.