Ultrarapid IHC to Detect Micrometastases Table 1. Procedures and time for immunohistochemical staining Routine IHC procedure Saving AC electric IHC procedure Rapid AC electric IHC procedure 135 Acetone fixation Washing with PBS Primary antibody Concentration of primary antibody Washing with PBS EnVisionTM+ System/HRP Mouse Washing with PBS 3,3'diaminobenzidine Approximate time required w/o AC electric field 60 min w/o AC electric field 30 min 4°C 10 min 5 min 3 times 5.0 µg/mL (1:200) 5 min 3 times 5 min 3 times 3 min 148 min anti-pancytokeratin antibody cocktail (AE1/AE3; PROGEN Biotechnik GmbH, Heidelberg, Germany) at 1:200 dilution (5 μg/mL) under a high-voltage (3.4 kV, offset 2.4 kV), low-frequency (18 Hz) AC electric field. The sections were then washed with PBS and incubated for 2 min with EnVisionTM+ System/HRP Mouse (DAB+) (Dako) under the same AC electric field. After again washing with PBS, the sections were developed with 3,3'diaminobenzidine sub- strate, counterstained with hematoxylin, dehydrated, and mounted on coverslips. Standard 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 10 min at 4°C, 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 sections were first incubated for 60 min with AE1/AE3 at 1:200 dilution (5 μg/mL). They were then washed with PBS, incubated for 30 min with EnVisionTM+ System/HRP Mouse (DAB+), washed again with PBS, developed with 3,3'diaminobenzidine substrate, counterstained with hemato- xylin, dehydrated, and mounted on coverslips. Saving 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, sections were incubated for 60–180 min with AE1/AE3 at 1:500–2,000 dilution (0.5–2.0 μg/mL) under a high voltage (3.4 kV, offset 2.4 kV), low frequency (18 Hz) AC electric field. The sections were then washed with PBS and incubated Room temperature 2 min 30 sec once AC electric field 60–180 min 0.5–2.0 µg/mL (1:500–2,000) AC electric field 2 min 30 sec once 30 sec once 3 min 69–189 min Room temperature 2 min 30 sec once AC electric field 2 min 5.0 µg/mL (1:200) 30 sec once AC electric field 2 min 30 sec once 3 min 11 min for 2 min with EnVisionTM+ System/HRP Mouse (DAB+) (Dako) under the same AC electric field. After washing again with PBS, the sections were developed with 3,3'diami- nobenzidine substrate, counterstained with hematoxylin, dehydrated, and mounted on coverslips. III. Results We initially visualized the mixing effect of our device by applying the high voltage (3.4 kV), low frequency (18 Hz) AC electric field used for IHC analysis to ferrite particles in PBS. We observed that the ferrite particles were well mixed within 9 sec (Fig. 1E). We then stained metastatic NSCLC cells in lymph nodes with an anti-pancytokeratin antibody cocktail, AE1/ AE3, using standard IHC procedures or rapid AC electric or saving AC electric IHC procedures. Tumor cells within lymph nodes were stained equally well using the rapid AC electric procedure or the standard IHC procedure. Moreover, when the AC electric field was applied to sections for 60 min, the concentration of AE1/AE3 could be diluted 1:500, and when the AC electric field was applied for 180 min, the AE1/AE3 could be diluted 1:2,000. Without the AC electric field, the sections were not stained with AE1/AE3 at this dilution. Figure 2 depicts typical images obtained using the standard, rapid AC electric, and saving AC electric IHC procedures (dilution 1:2,000, 180 min AC electric field), respectively. An essential purpose of IHC analysis during surgery for NSCLC is detection of sentinel node micrometastasis without misdiagnosis. This means that to apply the rapid AC electric field IHC procedure in practice, its ability to detect micrometastases must be at least equal to that of the standard IHC procedure. Therefore, to further determine the effec-