Fluorescence in situ hybridization (FISH) analysis

Prepared microscope slides were pretreated with RNase A and pepsin, then denatured with 70 % formamide/2xSSC at 72 °C, dehydrated in a series of cold ethanol washes and air-dried.

The BAC clone RP11-379 K17 encoding SEC62 (ImaGenes, Berlin, Germany) was biotin labeled using the BioPrime DNA Labeling System (Invitrogen, Life Technologies, Darmstadt, Germany). As internal control, a centromeric probe for chromosome 10 (D10Z3) labeled with digoxigenin by standard nick-translation according to the manufacturer’s instructions (Roche Diagnostics GmbH, Mannheim, Germany) was used. After probe hybridization overnight, the slides were washed two times in 2× SSC at 42 °C and three times in 50 % formamide/2× SSC at 42 °C. Immunofluorescence detection of the biotin signals was carried out using Streptavidin-FITC and -biotinylated anti-Streptavidin antibodies (Vector Laboratories, Burlingame, CA, USA). For the detection of the digoxigenin signals, anti-Dig-Cy3 and goat-anti-mouse-Cy3 (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) were used. The slides were mounted in an anti-fade solution containing DAPI (4, 6-diamidino-2-phenylindole; Vector Laboratories, Burlingame, CA, USA) and analyzed with the BX61 fluorescent microscope equipped with a charge-coupled device camera (Olympus, Hamburg, Germany). In total, 200 non-overlapping, morphologically well-preserved nuclei per slide were analyzed. Thereby, we selectively evaluated the number of FISH signals in the morphologically conspicuous nuclei in the CIN-I, CIN-II, CIN-III and SCC (histological diagnosis) cases. For the “no CIN” cases, every nucleus was considered. Gains were defined as three or four signals per probe; five or more signals were defined as amplification. The specificity of each probe was determined by hybridizing and enumerating normal human lymphocytes and metaphase spreads, prepared according to standard protocols, for cutoff ranges and an analysis of cross hybridizations by non-stringency of hybridization conditions.

FISH analyses were performed on cytological specimens in a representative subset of 20 patients with histological diagnoses of “no CIN” (n = 5; cytological diagnosis NILM [n = 5]), CIN-I (n = 5; cytological diagnosis ASCUS [n = 1], LSIL [n = 3] and HSIL [n = 1]), CIN-II (n = 5; cytological diagnosis ASCUS [n = 1] and HSIL [n = 4]), CIN-III (n = 5, cytological diagnosis HSIL [n = 4] and SCC [n = 1]) and SCC (n = 5; cytological diagnosis SCC [n = 5]).