Elipse e800

For each studied antibody, antigen expression evaluation was scored on a two-point scale: 0 - negative IHC reaction result, 1 – positive IHC reaction result. We consider such approach transparent and useful in routine diagnostics. Slides were examined in the light microscope ELIPSE E800 (Nikon Instruments Europe, Amsterdam, Netherlands) at 10x and 20x original objective magnification. Scoring was repetitively performed by three pathologists who were blinded to the clinical information.

The day prior to the experiment, 10⁵ U2OS cells were seeded onto 11 mm glass coverslips and allowed to attach overnight at 37 °C/5 % CO₂ in DMEM containing 10 % FBS (Gibco). Cells were treated with 100 μM sodium (meta)arsenite (Sigma Aldrich) for 1 h to induce the formation of stress granules and then with 4 % paraformaldehyde solution at room temperature for 15 minutes followed by blocking and permeabilization with 5 % normal horse serum, 0.1 % digitonin in Tris-buffered saline. Staining was performed with anti-eIF3b (Santa Cruz), anti-SK1-Hedls (Santa Cruz), and patient sera for 1 h at room temperature. Secondary antibodies (anti-goat-Cy3, anti-mouse-Cy2, and anti-human-Cy5) were purchased from Jackson Laboratories and incubated at room temperature for 1 h. Conventional fluorescence microscopy was performed using a microscope (model Elipse E800, Nikon, Tokyo, Japan) with epifluorescence optics with a digital camera (model CCD-SPOT RT; Diagnostic Instruments, Sterling Heights, MI). Images were compiled using Adobe Photoshop software (CS6; Adobe Systems, San Jose, CA).