Zen 2.3 pro software

Hep3B cells were plated at high density (500,000 cells/ml) in regular tissue culture 10 cm dish (for monolayer) or in low attachment flasks (for spheres) in complete MEM and cultured for 48hrs with a media change at 24hrs. The spheres were stained with Hoechst-33342 and imaged at 48hrs using Zeiss Observer.Z1 microscope equipped with Axiocam 503 mono (Zeiss) camera. The individual channel images of Hoechst-33342 were pseudo-colored to red, merged with bright field and exported using ZEN 2.3 Pro software (Carl Zeiss Microscopy, GmbH, 2011, Blue edition). The final composite was done using Adobe Photoshop CS5 (Adobe Systems Inc., San Jose, CA, USA). Similar experiments were performed to collect monolayer cells and spheres for RNA isolation for RT-PCR/qRTPCR analysis using Hep3B parental cells or miR-520G stably transfected cells.

The cells were grown onto a 15 mm coverslip (Chemglass Life Sciences) and treated with 1 μM CPT for 3 hr or 1 Gy X-ray (Faxitron CellRad) and 1 hr release. Then the cells were fixed with 4% formaldehyde in PBS for 20 min, rinsed with PBS and permeabilized with 0.5% Triton X-100 for 15 min. The cells were washed and blocked with 5% PBS/PBST for 30 min. Next, the cells were washed with PBST and incubated with anti-mouse AlexaFluor 488 (1:1000) for 45 min. After washing with PBST three times, the cells were stained with Vectashield mounting medium containing DAPI (Vector Laboratories). Images were captured by Axio Imager A2 at 63x magnification (Zeiss) and analyzed using Zen 2.3pro software (Zeiss). At least 184 cells were counted and the experiments were performed three times.

Images were acquired using Zen 2.3 pro software (Carl Zeiss AG, Feldbach, Switzerland) and processed using Image J 1.52n software (Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, MA, USA). Semi-quantitative data as a function of skin depth was extracted from processed images using the pixel count function in Image J 1.52n software.

For immunofluorescent staining, slides were left to dry at room temperature for 30 min and washed three times in PBS. Tissue sections were blocked with 10% NGS (Normal Goat Serum) in PBS. Anti-swine IgG Alexa Fluor 488 (1:100, Jackson ImmunoResearch Labotory, USA) was used to assess status of the Blood–Brain Barrier (BBB). After the blocking slide-mounted tissues were incubated with antibodies overnight in 4 °C. Next day antibodies were removed and sections were coversliped with antifade mounting medium containing DAPI (Fluoroshield, Sigma Aldrich, Germany). Images were captured using Zeiss Axio Observer microscope (Zeiss, Germany) and analyzed using ZEN 2.3 pro software (Zeiss, Germany). As our region of interest (ROI) we selected areas inside of the lesion (ipsilateral hemisphere and corresponding structure in the intact contralateral hemisphere. Tissue was outlined using a parametric active contour method (spline contour) and AF488 intensity mean values were measured.

All microscopy procedures, three-dimensional reconstructions, animations, and maximum intensity projections were performed as described previously (Kitaeva et al., 2016 (link); Ilina et al., 2018 (link)). Examination and imaging of fluorescent protein patterns were performed under a LSM 780 upright confocal laser scanning microscope (ZEISS, Germany) equipped with a Plan-Apochromat 20×/0.8 numerical aperture DICII objective and a Plan-Apochromat 40×/1.3 numerical aperture DICIII oil immersion objective. Samples were imaged with a 488 nm excitation laser line and an emission spectrum of 490–525 nm for both eGFP or mNeonGreen. For DAPI-stained nuclei, the 405 nm excitation laser line and an emission spectrum of 412–464 nm were used. A multitrack (line by line) scan mode was applied. The ZEN 2.3pro software (ZEISS) was used for image processing. At least 14 roots were used for each reporter assay. The distance from the initial cell to the first cell in file labeled with eGFP or NeonGreen in nuclei was measured in the ZEN software (ZEISS) after acquisition. Statistical analysis and graphical visualization were performed in SigmaPlot 12.5 (Systat Software, United States) using one-way analysis of variance (ANOVA) on ranks (Kruskal–Wallis).