Slices were washed 3 × 5 min in HBS-0.05% BSA, then four changes of distilled H2O for 2 hr, then incubated for 2 hr on a shaker in 0.5 ml of GoldEnhance EM (Nanoprobes) mixed according to manufacturer's directions.
Slices were washed 3 × 5 min in HBS-0.05% BSA, then fixed at room temperature in perfusion fixative for 20 min. After four changes of distilled H2O for 2 hr, slices were incubated for 3 hr on a shaker in 0.5 ml of GoldEnhance EM solution.
Approximately 1 mL of lipiodol (Ultrafluid; E-Z-EM Canada, Inc., Anjou, Quebec, Canada) was injected into the angiocatheter placed in the upstream popliteal prenodal vessel and the lymphatics/nodes were visualized using a mobile fluoroscopy system (BV Pulsera; Philips Healthcare, Andover, MA, USA).
These calibration standards typically contain 0.2 to 200 pg of each EM in 0.5 ml of MCF-10A cell lysate and were assayed in duplicate.
Working standards of EMs and SI-EMs at 8 ng/ml were prepared by dilutions of the stock solutions using methanol with 0.1% L-ascorbic acid.
Stock solutions of EMs and SI-EMs were each prepared at 80 μg/ml by dissolving 2 mg of the estrogen powders in methanol with 0.1% L-ascorbic acid to a final volume of 25 ml in a volumetric flask.
Immediately prior to scanning, patients were asked to drink approximately 500 700 ml of a water-based neutral contrast (VoLumen, EZ-EM) to distend the duodenum.
We routinely use gas distension of the stomach with 1 2 sachets of effervescent granules (EZ Gas II Effervescent Granule Packets, EZ EM, New York) administered with a small amount (5 10 ml) of water.
Then, 225 ml of low concentration barium (4.9%) and sorbitol-containing solution (E-Z-CAT, E-Z-EM, Inc, Quebec, Canada) at room temperature were added to this suspension.
0.5 ml of 5 µM Na-F (Sigma-Aldrich, Buchs, Switzerland) was added to the apical chamber and 1.5 ml EM (without phenol red) to the basal chamber.
