Application suite x las x software package

Generation of reactive oxygen species (ROS) in the control and PET NPs treated embryos was visualized in intact zebrafish embryos at 96 hpf using the probe chloromethyl-2′, 7′ dihydrodichlorofuorescein diacetate (CM-H₂DCFA; Molecular Probes), which is known as nonfluorescent cell-permeative compound. Intracellular esterases cleave the acetate groups such that the non-fluorescent dye 2′, 7′-dichlorofuorescein (DCF) is retained intracellular and oxidized by intracellular ROS and becomes fluorescent. For sample preparation, CM-H₂DCFA (1 mM solution in 4% DMSO) was added to control and PET NPs treated embryos in embryo medium to a final concentration of 10 μM, and incubated in the dark for 60 min at 28 °C. Subsequently, excess CM-H₂DCFA was removed by washing embryos 3 times with embryo medium. Subsequently, embryos were placed on a borosilicate glass coverslip and anesthetized with tricaine solution (200 mg/L) during fluorescence imaging. The fluorescence images of stained zebrafish embryos were captured using inverted laser-scanning confocal microscope (Leica DMi8 / TL LED, Leica Microsystems CMS GmbH). The fluorescent product DCF was detected with an excitation wavelength of 485 nm, and an emission wavelength of 530 nm, using a Leica HC PL Apo CS2 (5x/0.15 Dry) objective and Leica Application Suite X (LAS X) software package, version 3.1.5, to acquire images.

The uptake and distribution of OTA in the zebrafish embryos was visualized by laser-scanning confocal microscopy. Embryos, both untreated and treated (with 4.0 µM OTA), were washed with Milli-Q water, and placed on a borosilicate glass coverslip slide in a solution containing embryo medium^{80 (link)} with Tricaine anaesthetic (i.e., 1 mg/mL). After 3 min (to assure anaesthesia), images were captured using inverted laser-scanning confocal microscope (Leica DMi8/TL LED, Leica Microsystems CMS GmbH) with a 380-nm laser for excitation, and emission filter set to 460-nm band pass. Images were acquired with a Leica HC PL Apo CS2 (5x/0.15 Dry) objective, and Leica Application Suite X (LAS X) software package version 3.1.5. For quantitative analysis of OTA fluorescence, the images were exported and further analyzed in ImageJ software (ImageJ, USA). Using color deconvolution, the colors were unmixed, and the areas were selected, and subsequently, the number of particles were calculated by the Image-based Tool for Counting Nuclei. Any alterations in brightness and contrast were equally applied to the entire image set. The quantification of OTA fluorescence was performed, and data were exported to Origin Pro v. 8 software for further analysis.