Envision automated immunostainer

Slides were incubated with 3% peroxide-methanol at room temperature for 20 min to block endogenous peroxidase, and with 1% diluted goat serum to block nonspecific binding sites. ERα (Dako, Glostrup, Denmark) was added following the avidin-biotin-peroxidase procedure using an EnVision Automated Immunostainer (Dako). Slides were then counter-stained with Gill's hematoxylin (Santa Cruz, CA, USA). Negative control sections were treated as described above, but the primary antibody was replaced by 0.01 M phosphate-buffered saline.

All biopsies were fixed in 10% neutral buffered formalin and embedded in paraffin using conventional histopathological methods. Paraffin blocks were serially dissected at a thickness of 5 µm with ten histological sections preserved per glass slide. Each section was stained with hematoxylin and eosin (H&E) for routine microscopy, general neural marker protein gene product 9.5 (PGP9.5, Dako, Glostrup, Denmark) as well as a marker of vascular smooth muscle, smooth muscle actin (SMA; Dako), following the avidin-biotin-peroxidase procedure using an EnVision Automated Immunostainer (Dako). As negative controls, sections were treated in an identical manner but with 0.01 M phosphate-buffered saline replacing the primary antibody. All blind-coded sections were evaluated independently by two experienced pathologists and examined under a double-headed microscope (BH2; Olympus, Tokyo, Japan). Dropsical tissue would be excluded.
The exact numbers of nerves and vessels were manually quantified in five consecutive fields under high power (×40). Blood vessels were classified in four categories according to diameter: small artery (0.3–1 mm), small vein (≥200 µm), arteriole (<300 µm) and venule (<200 µm) [23] . Incomplete blood vessels on the right edge were quantified and those on the left were ignored.

Cells were cultured as described above, then fixed in 4% formaldehyde for 20 min and permeabilized with 0.3% Triton X-100 for 30 min. Three cases of normal endometrium(1: proliferative phase, 2: secretory phase) and three human endometrial carcinoma tissues of various differentiation grades for the comparison of the normal tissues, Grade 1, Grade 2 and Grade 3 tumors were obtained from Peking University First Hospital. The pathology diagnosis and tumor grade were determined by 2 staff pathologists in the Department of Pathology, Peking University First Hospital. We have retrospectively measured the expression level of NGAL in seventy-seven cases with endometrial carcinoma and twenty-eight cases with normal endometrium by western bloting [29 ]. Human endometrial cancer tissues were fixed in 10% formalin, embedded in paraffin, and sectioned. Endogenous peroxidase were blocked in 3% H₂O₂ for 10 min then sections were incubated with the primary rabbit antibody (anti-NGAL antibody, anti-E-Cadherin 1:100 and anti-Vimentin 1:100) following the avidin-biotin-peroxidase procedure using an EnVision Automated Immunostainer (Dako). Nuclei were stained with hematoxylin-eosin (H&E) for routine microscopy. The slides were examined under a light microscope (AH3-RFCA; Olympus, Tokyo, Japan).