Axiocam 305 mono camera

GPC4 expression was evaluated using fluorescence imaging as previously described (7). Specifically, cells transfected with the CRISPR control, CRISPR/Cas9 GPC4, overexpression control, or GFP-GPC4 vectors were washed extensively with phosphate buffer saline (PBS), followed by fixation with acetone in 2 min. The cells were then incubated for about 24 h with rabbit polyclonal anti-human GPC4 antibody (Catalog # PA5-115301; Invitrogen, Lund, Sweden). After thorough PBS washes, the cells were incubated with goat anti-rabbit IgG, Alexa Fluor™ 594 (cat# A-21208; Molecular Probes, Eugene, OR, USA) for 4 h. Nuclei were visualized through DNA staining with 4′,6-diamidino-2-phenylindole (DAPI). The anti-human GPC4 antibody was omitted in the controls. Analysis of the fluorescent images was accomplished using an AxioObserver fluorescence microscope from Carl Zeiss equipped with a 63X/1.25 Oil M27 EC Plan-Neofluar objective and Axiocam 305 mono Camera. All images were scanned using identical exposure settings. Multitrack acquisition and sequential excitation of fluorophores were used to minimize channel crosstalk. The entire slide was scanned during microscopy, and immunofluorescence images were captured at 20× and 60× magnification.

Fluorescence in situ hybridization (FISH) assay with a BCAS1 probe (RP11-705A3, red labeling) and a Chromosome 20 Centromere probe (CHR20-10-RE, green labeling) (Empire Genomics, Buffalo, NY, USA) was performed in a sample of each tumor type studied. After deparaffinizing and rehydrating 2.5 μm tissue section, manufacturers’ automated hybridization protocol by Empire Genomics was carried out using the HYBrite platform (Abbott Molecular, Des Plaines, IL, USA). Cell nuclei were counterstained with DAPI. Fluorescence was analyzed using an Axioscope 5 microscope equipped with Axiocam 305 mono camera (ZEISS). For each sample, more than 100 nuclei were counted. Aneuploidy was defined as a chromosome count that differed from the normal 2 n chromosome number.

Cells were plated in 96-well Clear Round Bottom Ultra-Low Attachment Microplate (Corning, Corning, NY, USA) at a density of 500 cells per well and pelleted at the bottom of the well. The following day, the cell culture medium was replaced with a solution of 2% v/v Geltrex™ LDEV-Free Reduced Growth Factor Casement Membrane Matrix (Gibco, Waltham, MA, USA) diluted in cell culture media. After 7 days of culturing, with regular replenishments of cell culture media, spheroid formation was monitored and measured with a ZEISS Axio Vert.A1 inverted microscope with Axiocam 305 mono camera (ZEISS, Oberkochen, Germany).