134 Toda et al. by the lack of morphological observation. To overcome that limitation, we have now developed a device that enables us to complete IHC analyses within 15 min using an alternating current (AC) electric field (patent pending). The aim of the present study was to evaluate the clinical signifi- cance, reliability, and sensitivity of the novel ultrarapid IHC method developed at our institute using this device. II. Materials and Methods Ten consecutive patients with NSCLC were enrolled in the study between July 2010 and August 2010 after obtaining signed informed consent. Surgically resected specimens were used under approval of the Institutional Review Boards at Akita University School of Medicine and University Hospital. After a preoperative evaluation, the patients were taken to an operating room, and the standard preparations were made for a thoracotomy, lung resection, and mediastinal lymph node dissection. Lymph nodes from each patient were used for this study. IHC procedures Tissue preparation Lymph nodes were surgically resected from patients with NSCLC. Immediately after removal, the nodes were embedded in O.C.T. compound (Sakura Finetek Japan Co., Ltd., Tokyo, Japan) and frozen for 30 sec in liquid acetone at −80°C in Histo-Tek Pino (Sakura Finetek Japan Co., Ltd., Tokyo, Japan) and then transferred to a cryostat (CM1900 Leica, Wetzlar, Germany). The device for IHC analysis (Fig. 1) We have developed a device that reduces the time required for IHC, as well as the amount of antibody required for these analyses. With this device, we can apply a high- voltage, low-frequency AC electric field to the sections. The resultant coulomb force stirs the antibody solution on the sections. To examine the mixing effect of the device, the AC electric field was also applied to ferrite particles (average diameter: 50 nm) in PBS. To prevent evaporation of the antibody solution, the device was equipped with a humidi- fier, and all incubations were carried out under a humidified atmosphere. With the humidifier, the amount of antibody solution remained unchanged, even after 3 hr of incubation. Rapid AC electric IHC procedure (Table 1) Serial frozen sections were cut at 5 μm and placed on slides, air-dried for 30 sec, fixed in acetone for 2 min at room temperature, air-dried for 15 sec at room temperature, and subjected to the staining procedure. No attempt was made to block endogenous peroxidase. For staining, the sec- tions were incubated for 2 min with a monoclonal mouse Fig. 1. New device for AC electric field IHC. Panel A depicts a schematic diagram of a device for AC electric field IHC analysis. Panel B depicts a typical oscilloscope trace of the voltage and frequency of the AC electric field. A high voltage (3.4 kV, offset 2.4 kV), low frequency (18 Hz) AC electric field was applied to the sections. Panels 1C and 1D show the device, which was equipped with a humidifier to prevent evaporation of the antibody solution. To visualize the mixing effect of the device, the AC electric field was also applied to ferrite particles (average diameter: 50 nm) in PBS. Note that the brown particles (red arrows) were unevenly distributed at 1 and 4 sec, but were well mixed and homogeneously distributed within 9 sec (Panel E). I.T.O. electrode, indium tin oxide glass plate electrode; ab, antibody.