Magnetically Promoted Rapid Immunofluorescence Staining for Frozen Tissue Sections

Abstract

Current immunohistochemistry methods for diagnosing abnormal cells, such as cancer cells, require multiple steps and can be relatively slow compared with intraoperative frozen hematoxylin and eosin staining, and are therefore rarely used for intraoperative examination. Thus, there is a need for novel rapid detection methods. We previously demonstrated that functionalized fluorescent ferrite beads (FF beads) magnetically promoted rapid immunoreactions. The aim of this study was to improve the magnetically promoted rapid immunoreaction method using antibody-coated FF beads and a magnet subjected to a magnetic field. Using frozen sections of xenograft samples of A431 human epidermoid cancer cells that express high levels of epidermal growth factor receptor (EGFR) and anti-EGFR antibody-coated FF beads, we reduced the magnetically promoted immunohistochemistry procedure to a 1-min reaction and 1-min wash. We also determined the optimum magnetic force for the antibody reaction (from 7.79 × 10−15 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 cancer lymph nodes with anti-pan-cytokeratin antibody-coated FF beads to validate the utility of this system in clinical specimens. Under optimal conditions, this ultra-rapid immunostaining method may provide an ancillary method for pathological diagnosis during surgery. (J Histochem Cytochem 58:XXX–XXX, 2010)

Application of Magnetic Nanoparticles for Rapid Detection and In Situ Diagnosis in Clinical Oncology

“…Thus, there have been attempts to shorten the time of the procedure using ultrasound [ 72 ] and microwaves [ 72 , 73 ] that accelerate the antigen–antibody reaction with a stirring effect in addition to Brownian motion. Alternatively, Onishi et al used FF beads to develop MRIF staining, which shortens reaction and washing times using a magnet [ 26 , 74 ]. MRIF can be performed in two steps without secondary antibody, signal amplification, or DAB staining: (1) incubation with antibody-coated FF beads and (2) washing, because the antigen–antibody complex can be directly observed using a fluorescence microscope to observe the fluorescent material encapsulated in the FF beads ( Figure 6 ).…”

“…MRIF can be performed in two steps without secondary antibody, signal amplification, or DAB staining: (1) incubation with antibody-coated FF beads and (2) washing, because the antigen–antibody complex can be directly observed using a fluorescence microscope to observe the fluorescent material encapsulated in the FF beads ( Figure 6 ). This procedure reduces the time to a 1 min reaction and 1 min wash step with a magnet when applied to frozen sections of xenografted samples of A431 human epidermoid cancer cells that express high levels of epidermal growth factor receptor (EGFR) and anti-EGFR antibody-europium encapsulated FF beads ( Figure 7 A) [ 74 ].…”

“…The magnetic force (F) acting on an FF bead was calculated as F = −∇(− m b ⋅ B ), where m b is the magnetic moment of the FF beads, and B is the magnetic field strength of the magnet [ 75 ]. The distribution of the magnetic force was stronger at the margins than at the center of the magnet; therefore, we decided to agitate the magnet to obtain uniform staining [ 74 ]. The optimal distance between the A431 xenograft samples and the magnet using anti-EGFR antibody-coated FF beads for a 1 min incubation was within 2–5 mm, whereby the magnetic force =7.79 × 10 −15 N to 3.35 × 10 −15 N. A distance shorter than 2.0 mm showed unwanted background staining, and a distance greater than 5 mm showed insufficient staining.…”

“…The positive rate of conventional immunostaining was 96.5% (276/286) and that of MRIF was 92.7% (265/286). The coincidence rate was 94.8% (271/286) [ 74 ]. Normal tissue (i.e., breast tissue, tonsil, and lymph nodes) was analyzed.…”

SCARA5 suppresses the proliferation and migration, and promotes the apoptosis of human retinoblastoma cells by inhibiting the PI3K/AKT pathway

“…Immunofluorescence assay. Immunofluorescence assays were performed as previously described (18). Briefly, 1x10 3 cells were plated onto slides that had been treated with polylysine and were incubated at 37˚C for 4 h. Subsequently, the cells were fixed with 10% formaldehyde and then blocked with 5% bovine serum albumin (cat.…”