R catheter (Labotect, Goettingen, Germany). Then, the cavity was flushed using PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/20460822 3 mL of saline. The two solutions were mixed for analysis. On day 1 of the operation, blood was aspirated to examine the serum concentration of estradiol and progesterone to evaluate the phase of the menstrual cycle. A sample from endometrial curettage was also formalin-fixed and paraffin-embedded (FFPE) for a histological evaluation. Menstrual cycle can be dated into 6 sub-phases according to the anatomical changes within the endometrial biopsy including, early proliferative (EP) (days 5-7), mid-proliferative (MP) (days 8-10), late proliferative (LP) (days 11-14), early secretory (LS) (days 14-21), mid-secretory (MS) (days 22-24), and late secretory (LS) (days 25-28) phases [18]. Informed consent for all samples was obtained from all patients.To assess the specificity of the antibody, 400 ng of recombinant glutathione S-transferase (GST)-human SERPINE2 protein (Abnova, Taipei, Taiwan) was resolved by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) on a 10 gel slab (8.2 ?7.3 ?0.075 cm) and was transferred to a nitrocellulose membrane for immunostaining according to a previously described method [19]. Membranes were blocked with 10 (w/v) skim milk in phosphate-buffered saline (PBS) (blocking solution) for 2 h, and then incubated with our homemade anti-mouse SERPINE2 antiserum (1: 5000) [17,20], anti-human SERPINE2 antibody (1: 1000, catalog no. AF2980, R D Systems, Minneapolis, MN, USA), or another anti-human SERPINE2 antibody (1: 1000, product no. H00005270-B01, Abnova) in blocking solution for 2 h at 37 . After gentle agitation in four changes of PBS for 15 min each, PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/3081428 membranes were immunoreacted with horseradish peroxidase (HRP)-conjugated goat antirabbit immunoglobulin G (IgG) (1: 5000, GE Healthcare Life Sciences, Piscataway, NJ, USA), donkey anti-goat IgG (1: 3000, Jackson ImmunoResearch, West Grove, PA, USA), or horse anti-mouse IgG (1:3000, Cell Signaling Technology, Beverly, MA, USA), respectively, in blocking solution for 2 4,4,5,5-Tetramethyl-2-(2-methylprop-1-en-1-yl)-1,3,2-dioxaborolane h at 37 . After gentle agitation as mentioned above, immunoreactive bands were revealed using an enhanced chemiluminescent substrate according to the manufacturer's instructions (Western Lightning; PerkinElmer, Boston, MA, USA). To evaluate the sensitivity of the antibody, 100 g of a tissue extract from endometrial curetting was resolved, transferred onto a blot, and detected using anti-mouse SERPINE2 antiserum (1: 5000) or an anti-human SERPINE2 antibody (1: 1000) following the method described above. To examine the expression of SERPINE2 in the uterine fluid, 50 g of uterine luminal proteins was applied and detected using anti-mouse SERPINE2 antiserum (1: 3000) following the same method.Lee et al. Reproductive Biology and Endocrinology 2011, 9:38 http://www.rbej.com/content/9/1/Page 3 ofImmunohistochemical staining and signal analysisImmunohistochemical analysis was performed on an automatic staining machine (BenchMark XT, Ventana Medical Systems, Tucson, AZ, USA) using the iVIEW 3, 3-diaminobenzidine (DAB) detection kit (Ventana Medical Systems). After tissue sections (4 m) on slides were deparaffinized and hydrated, they were heated to 95 for 3-Bromo-5-chloro-2-fluoroaniline 8 min and then 100 for 4 min to induce antigen retrieval using Ventana Cell Conditioner 1 (Ventana Medical Systems). After cooling to room temperature for 30 min, the slides were treated with an iVIEW inhibitor at 37 for 4 min to inactivate the endogenous peroxidas.