Fluorescein isothiocyanate conjugated secondary antibody

For the immunofluorescence, heart tissues were fixed in a 10% buffered formaldehyde solution and embedded in paraffin. Paraffin was removed using a xylene wash, then heat-induced antigen retrieval was performed. The sections were then blocked with a 10% goat serum in phosphate-buffered saline for 1 h. The primary antibody anti-caspase-3 (1:200) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was incubated overnight at 4 °C. The sections were then incubated with a fluorescein-isothiocyanate-conjugated secondary antibody (1:500) (Sigma-Aldrich, St. Louis, MO, USA). Samples were visualized with a fluorescence microscope at 400× magnification (CIA-102; Olympus, Tokyo, Japan).

To document changes at tissue level in the expression of GLUT-1, sections of tumors from all experimental groups were exposed to immunofluorescent labeling using mouse monoclonal anti–GLUT-1 antibody (AbCam) followed by fluorescein isothiocyanate–conjugated secondary antibody (Sigma-Aldrich). Neoplastic expression of GLUT-1 was ascertained by the simultaneous detection of CD44. Briefly, GLUT-1-stained sections were incubated with rat monoclonal anti-CD44 antibody (HCAM-IM7, Santa Cruz Biotechnology, Dallas, TX) followed by specific Cy3-conjugated secondary antibody (1:50 Jackson ImmunoResearch Laboratories, West Grove, PA). Nuclei were counterstained with 4,6-diamino-2-phenylindole (DAPI). On sections from each experimental group, images of representative microscopic fields covering a tissue area of a minimum of 9.6mm² to a maximum of 12.4mm² were acquired with precalibrated gain and exposure time at 100x magnification using a fluorescence microscope (Olympus BX60).
The fractional area occupied by GLUT-1^pos/CD44^pos cells was evaluated by computing fluorescent signals using a software for image analysis (Image Pro Plus 4.0, Media Cybernetics, Inc., USA).

For immunofluorescence, tissues were fixed in 10% buffered formaldehyde solution and embedded in paraffin. Sections underwent microwave antigen retrieval, were blocked with 10% serum in phosphate-buffered saline, and were incubated with anti-caspase-3 (1:200) (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Binding sites of the primary antibody was revealed using a fluorescein isothiocyanate-conjugated secondary antibody (1:500) (Sigma–Aldrich, St. Louis, MO, USA). Samples were visualized with a fluorescence microscope at 400× magnification (CIA-102; Olympus).

For immunofluorescence assay, tissues were fixed in 10% buffered formaldehyde solution and embedded in paraffin. Sections were underwent microwave antigen retrieval, blocked with 10% goat serum in phosphate buffer saline, and incubated with polyclonal rabbit anti-caspase-3 antibody (Cell Signaling Technology). The primary antibody bound sites were visualized with fluorescein isothiocyanate conjugated secondary antibody (Sigma Aldrich) for a fluorescence microscopic observation at 400x magnification (CIA-102, Olympus, Tokyo, Japan) [3 (link), 21 (link)].

Cells were trypsinized, washed with ice-cold PBS, and fixed at a concentration of 2 × 10⁶ cells/mL in ice-cold 70% ethanol. For γH2AX analysis, samples were incubated with a mouse anti–γH2AX-specific antibody (clone JBW301; Millipore, Burlington, MA, USA) overnight at 4 °C, followed by incubation with a fluorescein isothiocyanate–conjugated secondary antibody (Sigma-Aldrich), as previously described [37 (link)]. For quantification of γH2AX positivity, a gate was arbitrarily set on the control, untreated sample to define a region of positive staining for γH2AX of approximately 5%. This gate was then overlaid on the treated samples. Samples were stained with propidium iodide to measure total DNA content and analyzed on a FACScan flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) with FlowJo software (Tree Star, Ashland, OR, USA).