Axiovision software 4

Fluorescent microscopy was used for live-screening of embryos/larvae at 24, 48, 72, 96 and 120 hpf. The number of thrombocytes in the caudal hematopoietic tissue (CHT) of zebrafish embryos/larvae was recorded through the detection of GFP-labeled thrombocytes. Images were captured with a Zeiss Axiocam HR camera using AxioVision software 4.8 (Carl Zeiss, Jena, Germany). Pixel intensity of fluorescent pictures was measured using ImageJ software (http://rsbweb.nih.gov/ij/).

After fixation, cells were placed into −20 °C acetone for 5 min for permeabilization, then rinsed with chilled PBS and incubated at room temperature with 1% BSA (Carl Roth, GmbH, Karlsruhe, Germany) in PBST (PBS + 0.1% v/v Tween-20 and including 0.3 M Glycine). Cells were incubated over night with the primary antibody (anti-α-Actinin 1:800 (ACTN2) clone EA-53, Sigma) for cell size analysis and identification of cardiomyocytes or anti-Orai1 (#1003, 1:200) to detect Orai1 proteins [50 (link)] at 4 °C. Afterwards, cells were washed three times with chilled PBS. Incubation with the secondary antibody (1:200 either anti-rabbit AlexaFluor488 or anti-mouse AlexaFluor594, Thermo Fisher Scientifics/Invitrogen) was done for 2 h for anti-Orai1 and for 1 h for all other antibodies. Nuclei were stained using DAPI (4′,6-diamidino-2-phenylindole, Thermo Fisher Scientifics/Invitrogen) (1.5 µg/mL in PBS for 5 min). During incubation times, the cells were protected from light to avoid fading. Afterwards, cells were mounted and covered in anti-fade (6 g Glycerin, 2.4 g Mowiol 4–88, 6 mL ddH₂O, 12 mL Tris-HCl 0.2 M pH 8.5, and DABCO 25 mg/mL solution). Cell size was analyzed after staining using the α-Actinin antibody under a fluorescence microscope (Z1, Zeiss) using the quantification tool of the AxioVision software 4.8 (Zeiss).

The migratory ability of GBM cells was determined by a scratch test. Briefly, cells were cultured in 6-well plates overnight, and then scraped with a micropipette tip to create gaps (wounds). Twelve hours later, employing an Axio Observer inverted fluorescence microscope (Carl Zeiss MicroImaging GmbH, Göttingen Germany) to image the scratched cell monolayers at 100× magnification, the average width of the gap that cells had migrated into was calculated via AxioVision software 4.8 (Carl Zeiss, Göttingen, Germany). The average width of each gap was calculated by the top, middle and bottom positions in the microscopic field.

Determination of nuclear position and cross wall orientation of cells was carried out using an AxioImager.Z1 Apotome microscope (Zeiss, Jena, Germany) with a ×20 objective and digital image acquisition controlled by AxioVision Software 4.8 (Zeiss). For examination of the mitotic spindle, confocal z-stacks were recorded at day 1 after subcultivation in the AtTUB6–GFP tobacco BY-2 strain using a Zeiss Observer.Z1 microscope with a Yokogawa CSUx1 detection system comprising a ×63 LCI-Neofluar Imm Corr DIC objective (NA 1.3) and the 488nm emission line of the Ar–Kr laser, as well as a spinning-disc device (YOKOGAWA CSU-X1 5000). Analysis of KCH binding on MTs under parthenolide treatment was performed using the same settings. Images were recorded at day 3 after subcultivation under treatment with 10 μM parthenolide. Untreated OsKCH cells at day 3 served as a control.

Spheroid outgrowth assays in HUVEC were performed as described in^{87 (link)}. Briefly, spheroids were generated by making drops containing 400 HUVECs in a methyl cellulose (20%) (Sigma-Aldrich, M-0512)/culture medium (80%) mixture onto a square petri dish (Greiner Bio-One, 688102). The dish was incubated overnight in upside down direction. Afterwards, spheroids were washed gently with PBS and resuspended in a methyl cellulose (88%)/FCS (12%) mixture and embedded in collagen type I (Corning, 354236) with Medium 199 (Sigma-Aldrich, M0650−100ML). Stimulation of Spheroids was performed with VEGF-A 165 (1 ng/mL) or bFGF (3 ng/mL) for 16 h. Images were generated with an Axiovert135 microscope (Zeiss). Sprout numbers and cumulative sprout lengths were quantified by analysis with the AxioVision software 4.8 (Zeiss).

For TdT-mediated dUTP-biotin nick end labeling (TUNEL), eyes were fixed in 4% paraformaldehyde (Carl Roth) for 4 h and embedded in paraffin, which is in accordance with standard procedures. After preparation of meridional sections, the TUNEL assay was performed in accordance with the manufacturer’s instructions (Deadend Fluorometric TUNEL system, Promega, Madison, WI, USA), mounted in fluorescent mounting medium containing 1:10 DAPI (Sigma-Aldrich, St. Louis, MO, USA) and analyzed on an Axio Imager fluorescence microscope with an integrated apotome module using the Axiovision software 4.8 (Carl Zeiss AG). For quantification, the number of apoptotic cells in the retinal ganglion cell layer and inner nuclear layer was counted and calculated as the number of TUNEL positive cells per 1000 µm using the Axiovision software 4.8.