Slide scanner

Tissue sections were dewaxed and rehydrated through a descending alcohol gradient, then immersed in boiled 10 mM sodium citrate, 0.005% Tween 20, pH 6.0 for 20 min to expose antigen sites. Endogenous peroxidase activity was quenched with H2O2 before staining with primary antibody (diluted in PBS with 0.1% Triton-X 100 and 3% goat serum) overnight at 4 °C in a humidified chamber, then biotinylated secondary antibody was applied (Vector Laboratories, CA, USA) for 2 h at room temperature (RT), and streptavidin-conjugated-Horseradish Peroxidase (Sigma, MO, USA) was added for 1 h. Chromogenic detection was with 3,3-diaminobenzidine (DAB) in the presence of H2O2 for 5 min, slides were counterstained for 2 min in Methyl Green (Sigma, MO, USA), washed in H₂O or water dehydrated and mounted in DPX mounting medium. The images were viewed using an Olympus Slide Scanner.

Hearts were perfused ex vivo via the aorta with 0.9% NaCl before fixation in 3.7% formaldehyde solution overnight. After embedding in paraffin, 5 μm sections were cut and stained for 60 min at room temperature in Sirius Red solution, i.e. 0.1% Direct Red 80 (Sigma, Taufkirchen, Germany) in saturated aqueous picric acid (Morphisto, Frankfurt, Germany) adjusted to pH 2.0 with sodium hydroxide [24 (link)]. The slides were tipped four times in 0.5% acetic acid solution and incubated 5 min each in ethanol and in isopropyl alcohol. After two further incubations for 10 min in xylole, the slices were embedded in Histokitt (Carl Roth) and recorded at 20x magnification using a slide scanner (Olympus, Hamburg, Germany). The extent of fibrosis based on the amount of collagen was quantified in two complete heart sections using cellSens Dimensions software (Olympus) by a scientist blinded to the genotype of the mice.
Kidneys were immersion-fixed in 3.7% formaldehyde solution overnight and embedded in paraffin. Immunohistochemical demonstration of eGFP was performed on 5 μm sections using a rabbit anti-eGFP polyclonal antibody (Abcam, Cambridge, UK) and the Vectastin ABC kit (Vector Laboratories, Burlingame, CA) using DAB (3,3’-diaminobenzidine tetrachloride) as a substrate. Quenching of endogenous peroxidase activity was achieved by treating the sections with 3% H₂O₂ in ethanol.

A total of 1 × 10⁵ cells were seeded in 6-well plates with a coverslip at the bottom of each well. Cells were left to attach overnight and then treated with cisplatin at the respective LD₅₀ concentration. After 72 hours, surviving cells were collected at 0, 5, and 10 days. Wells were washed with PBS and 1 mL of methanol:acetone (1:1), after which the plates were frozen overnight at −20°C. A total of 1 mL/well Giemsa (Merck, #48900) was added and for a following 1-hour incubation, the wells were washed three times with PBS. Coverslips were then mounted and imaged using slide scanner (Olympus).

A portion of tissue sample was fixed in 4% paraformaldehyde in PBS before processing overnight using an automated tissue processor (Shandon Citadel 2000). Samples were embedded in molten paraffin wax using a Leica EG1150H embedding station, sectioned at 3 μm, and mounted onto positively charged slides using a Leica RM 2155 microtome. Sections were dewaxed, rehydrated through a descending alcohol gradient into distilled water, then stained with H&E using a Thermo Fisher Scientific Shandon Varistain 24–4 autostainer, then mounted in DPX mounting medium (Sigma, MO, USA) and left to dry overnight before imaging using an Olympus Slide Scanner.

Images of BTIC cultures were captured using a Zeiss Axiovert 40 CFL inverted microscope and AxioVision software or the IncuCyte Zoom (Essen Bioscience). An Olympus Slide Scanner was used to image the brain sections at the University of Calgary Hotchkiss Brain Institute Core Facility. OlyVia (Olympus Life Science) software was used to analyze the images. Statistically significant differences between control and treated BTIC groups were evaluated by means of analysis of variance (ANOVA). Data are illustrated in bar graphs, including mean ± SEM. Asterisks denote statistical significance. For Kaplan-Meier survival studies, statistical difference in median survival was determined by the log-rank test. For in vivo microsomal stability analyses, data are illustrated in bar graphs, including mean ± SD. All analyses were performed using GraphPad Prism Version 6.0.