Choi YJ, Yun HR, Yoo KE, Kim JH, Nam SJ, Choi YL, Ko YH, Kim BT, Yang JH

Choi YJ, Yun HR, Yoo KE, Kim JH, Nam SJ, Choi YL, Ko YH, Kim BT, Yang JH. Intraoperative examination of sentinel lymph nodes by ultrarapid immunohistochemistry in breast cancer. anti-EGFR antibody-coated FF beads, we reduced the magnetically promoted immunohistochemistry procedure to a 1-min reaction and 1-min wash. We also decided the optimum magnetic pressure for the antibody reaction (from 7.79 10?15 Pemetrexed disodium N to 3.35 10?15 N) and washing (4.78 10?16 N), which are important steps in this technique. Furthermore, we stained paraffin-embedded tissue arrays and frozen sections of metastatic breast malignancy lymph nodes with anti-pan-cytokeratin antibody-coated FF beads to validate the power of this system in clinical specimens. Under optimal Des conditions, this ultra-rapid Pemetrexed disodium immunostaining method may provide an ancillary method for pathological diagnosis during surgery.?(J Histochem Cytochem 58:XXXCXXX, 2010) Keywords: cancer, fluorescent ferrite beads, immunostaining, intraoperative diagnosis, magnetic field Introduction Immunohistochemistry (IHC) staining is widely used for diagnosing abnormal cells such as those found in cancerous tissue. Antibodies bind specifically to antigens in biological tissues, and IHC staining can be accomplished by a number of methods. The avidin-biotin complex method is one of the most commonly used IHC staining systems, which involves four sequential actions: (1) primary antibody, (2) biotin-labeled secondary antibody, (3) avidin-biotin-peroxidase complex, and (4) diaminobenzidine (DAB) staining. Hybridization of the primary antibody to the antigen is typically accomplished with conditions that range from overnight at 4C to 15 min at temperatures as high as 37C; conditions are largely dependent on primary antibody affinity.1 In clinical practice, intraoperative frozen sections are usually examined with hematoxylin and eosin (HE) staining. To detect small numbers of tumor cells in sentinel lymph nodes or surgical margins, intraoperative IHC staining is usually desired.2 To complete the staining during surgery, a number of rapid IHC methods have been reported to reduce the reaction time of these processes.3C16 For example, ultrasound irradiation shortens the antibody reaction time to 1 1.5 Pemetrexed disodium min,3 while Hatta et al.3 reported that in addition to Brownian motion, stirring increased the stochastic collision between antibodies and antigens to enhance bond formation. Other rapid IHC systems also reduce the staining time with a stirring effect created using external energy such as microwaves4,6,7 or an alternating current electric field,13,14,17 reducing the method to <20 min (Table 1). However, these methods are indirect and require many actions including secondary antibodies and washing. Nevertheless, a direct method for decreasing the overall reaction time using a fluorescently labeled primary antibody or premixing the primary antibody and secondary antibody omitted the signal amplification process using a secondary antibody (Table 1). Table 1. Current Rapid Immunostaining Protocol for Frozen Sections. is shown in Fig. 3A to ?toC.C. To calculate the magnetic Pemetrexed disodium pressure is the distance from the surface of the magnet. The magnetic strength at various distances above the magnet are shown (left: = 2.0 mm, center: = 5.0 mm, right: = 11 mm). The correspondence between the color and pressure (distance, Y: log (Fabs) [log (is the distance from the element and 0 is the magnetic permeability in a vacuum. The magnetic flux density of the cylindrical magnet was calculated by the summation of Ba. The distribution of the magnetic pressure was affected by the distance from the magnet and was stronger at the margins than at the center of the magnet. The magnetic moment (m) of a magnetized FF bead is usually shown in Fig. 3D. The mass ratio of ferrite and polymer was 1:2, the mass densities of ferrite and polymer were 5.0 and 1.1 g/cm3, respectively, and the diameter of the FF bead was 200 nm. We obtained the mass of a bead, which was approximately 6.23 10?15 g, and the magnetic moment of an FF bead. Therefore, the magnetic pressure (F) acting on an FF bead was calculated as follows: F=mx(dBdx)+mz(dBdz), assuming the uniform magnetization of the FF beads and axial symmetry. Antibody-coated FF Beads and Evaluation of the Stained Images Before magnetic immunofluorescence staining, the amount of anti-EGFR antibodies immobilized on FF beads was 59.8 g/mg beads, as calculated using the bicinchoninic acid protein assay.27 The results of MRIF staining of A431 cells by anti-EGFR antibody-coated FF beads are shown in Fig. 4A. Vivid red fluorescence was observed in the A431 cells treated with anti-EGFR antibody-coated FF beads, indicating that EGFR was specifically stained by MRIF using anti-EGFR antibody-coated FF beads with a magnet. To ensure the specificity of the technique, a swine superficial lymph node was used as a negative control. Only a small number of fluorescent particles were observed in the lymph node in which EGFR was not expressed (Fig. 4B). Fluorescence was negligible in the A431 cells treated with IgG control antibody-coated, antibody-uncoated.