Sections were slightly counterstained with Mayer’s hematoxylin and mounted in aqueous mounting medium (Glicergel, Dako). Dako control slides were used as positive controls and the negative control was performed by omitting the application of the primary antibody. IHC scoring was based on the membrane immunoreactivity, according to the American Joint

Committee [17]: 0, no reactivity, 1+, weak reactivity, 2+, moderate reactivity, 3+, strong reactivity. Chromogenic in situ hybridization Formalin fixed paraffin embedded (FFPE) sections were deparaffinized, dehydrated, AG-881 molecular weight air dried, and heated in boiling tissue heat pre-treatment buffer for 15 minutes using a SPoT-Light® FFPE reagent kit (Zymed, Histoline, Milan, Italy). Enzymatic digestion was performed using SPoT-Light® FFPE digestion enzyme (Zymed) for 2-3 minutes at RT. After dehydration, histological slides were air dried and the ready-to-use double-stranded DNA digoxygenin-labelled EGFR probe (Zymed) or the biotin labelled chromosome 7 centromeric probe (Zymed) were applied. Denaturation was performed by incubating the slides, covered with a CISH cover-slip, on a 96°C heating block for 5 minutes, and hybridization was performed by placing the slides in a humidity chamber at 37°C overnight. After removing the cover-slips, a stringent wash was performed in 0.5× saline-sodium citrate buffer

at 80°C for 5 minutes. The endogenous peroxidase activity and unspecific staining were blocked AZD5363 mw by applying 3% hydrogen peroxide and the CAS-Block™, respectively.

A mouse antidigoxygenin antibody was added to the slides hybridized with EGFR probe for 45 minutes at RT followed by incubation with a polymerized peroxidase-goat anti-mouse antibody (Dako) for 45 minutes at RT. On the FFPE tissue slides, the colorimetric signal of chromosome 7 centromeric probe was improved by incubating RG7420 chemical structure the slides with a mouse antibiotin antibody (Dako) for 45 minutes at 37°C. A DAB chromogen substrate system was used to generate a sensitive signal that could be viewed with a Nikon ECLIPSE 55i transmission light-brightfield microscope (Nikon, Amstelveen, The Netherlands) after Mayer’s haematoxylin counterstaining. Fluorescence in situ hybridization FISH was performed using the LSI EGFR (SpectrumOrange™), a locus-specific probe for the EGFR human gene locus (7q12) and the chromosome enumeration probe (CEP 7, SpectrumGreen™) for alpha-satellite DNA Vistusertib solubility dmso located at the centromere (7q11.1-q11.1) (Vysis, Inc., Downers Grove, IL). The assay was carried out according to the manufacturer’s instructions. Shortly after deparaffinization, the FFPE specimens were incubated in the pre-treatment solution (82°C, 30 minutes) and then digested with protease (37°C, 15 minutes). After washing, the slides were counterstained with 4′,6-diamidino-2 phenylindole (DAPI) and analyzed using a fluorescent microscope. An average of 30 nuclei was counted for each case.