Dfc450 c camera

At 25 hpf, 10 zebrafish embryos per Petri dish (n = 30 per experimental condition) were randomly selected. The number of complete body contractions each zebrafish made in 30 second period was counted and was used as indicative of motor neuron activity [27] (link). Representative videos of each experimental condition were taken using Leica Application Suite (Leica Microsystems Ltd) and a Leica MZFLIII microscope (Leica Microsystems Ltd) equipped with a DFC450C camera (Leica Microsystems Ltd).
For the analysis of the touch-evoked escape behaviour, 10 zebrafish larvae per Petri dish (n = 30 per experimental condition) were randomly selected. Touch-evoked escape behaviours were elicited by touching a larva in the tail up to 6 times with a pair of forceps at 5 dpf. Three categories were distinguished, responders, late responders and non-responders, to which the following scores were given: 3 points for responders: fish quickly react (swimming or flicking the tail) to the stimuli after 1 or 2 twitches; 2 points for late responders: fish react (swimming or flicking the tail) to the stimuli after 3, 4 or 5 twitches; and 1 point for non-responders: fish do not react to the stimuli after more than 5 twitches. Representative videos were recorded with the system described above.

Visualization of male meiotic chromosomes and of female sporogenesis in Ranunculus samples was carried out with a Leica microscope DM5500B. Images of the orcein-stained PMCs and the cleared ovaries were taken with a DFC 450C camera and LAS V41 software (Leica Microsystems, Wetzlar, Germany). For fluorescent picture imaging, the same microscope equipped with the DFC 365FX camera, the FLUO-filter cube A4 and the LAS AF 3.1.0 software was applied (Leica Microsystems CMS GmbH, Wetzlar, Germany).

For cochlear and middle ear examination, mice were euthanized by intraperitoneal injection of 400 mg/kg ketamine and 80 mg/kg xylazine. After cessation of breathing, PBS was perfused through each animal via a cannula inserted into the left ventricle for 5 min, followed by 10% neutral buffered formalin for 5 min. Cochleae were dissected from the temporal bones and post-fixed for 1 hr at room temperature. Cochleae were washed in tris-buffered saline and decalcified in 10% EDTA for 5 days at 4°C with gentle rolling. Cochleae were oriented in 1% agarose in PBS in 10 mm×10 mm×5 mm cryomolds (Sakura Finetek, Torrance, CA, USA) and paraffin-embedded. 2 µm sections were cut parallel to the modiolus using a microtome and stained with hematoxylin and eosin (H&E). Sections of middle ear cavity were also examined for otitis media.
For eye examination, mice were euthanized via cervical dislocation and eyes fixed in 10% neutral buffered formalin before being embedded in paraffin. 2 µm sagittal sections were cut using a microtome and stained with H&E. Sections were imaged with a DM1000 compound microscope (Leica Microsystems, North Ryde, Australia) and DFC450 C camera (Leica Microsystems).

Cells grown on all samples were examined by means of fluorescence imaging. After 24 h and 72 h of culture, SaOs2 cells were fixed with 4% paraformaldehyde at room temperature and kept in phosphate-buffered saline (PBS) at 4 °C before labeling. The fixed cells were then permeabilized with 0.2% TritonX-100 and blocked in 0.5% bovine serum albumin. In order to visualize the actin filaments, the cells were stained with Alexa Fluor 488-conjugated Phalloidin (Cell Signaling, Technology, Danvers, MA, USA). The cells were then treated with 1 μg/mL Hoechst (Cell Signaling, Technology, Danvers, MA, USA) in order to label the nuclei. After each incubation, the samples were washed three times with PBS. In the end, the specimens were analyzed on glass slides using a DM 4000 B LED fluorescence microscope equipped with a DFC 450 C camera (Leica Microsystems, Wetzlar, Germany) with appropriate filters.

Mice were euthanized by intraperitoneal injection of 400 mg/kg body weight ketamine and 80 mg/kg body weight xylazine. After cessation of breathing, PBS was perfused through each animal via a cannula inserted into the left ventricle for 5 minutes, followed by 10% neutral buffered formalin for 5 minutes. Cochleae were dissected from the temporal bones and post-fixed for 1 hour at room temperature. Cochleae were washed in tris-buffered saline and decalcified in 10% EDTA for 5 days at 4°C with gentle rolling. Cochleae were oriented in 1% agarose in PBS in 10 mm×10 mm×5 mm cryomolds (Sakura Finetek, Torrance, CA) and paraffin-embedded. 2-μm sections were cut parallel to the modiolus using a microtome and stained with hematoxylin and eosin (H&E). Sections were imaged with a DM1000 compound microscope (Leica Microsystems, North Ryde, Australia) and DFC450 C camera (Leica Microsystems).

To test for sexual versus apomictic embryo sac development, a total of 338 spikelets from 16 different individuals was analyzed by microscopic observation of megasporogenesis and embryo sac development following methods described in Young et al. [61 ]. Inflorescences that were previously fixed in FAA and stored in ethanol 70% were dehydrated in 100% ethanol for 30 minutes. Afterwards, they were incubated in 300 μl of upgrading series of methyl salicylate (Merck KGaA, Darmstadt, Germany) diluted in ethanol (25%, 50%, 70%, 85%, and 100%) for 30 minutes in each steps. Spikelets were dissected to prepare the ovules and anthers, then ovules and anthers were amounted in methyl salicylate on glass slides. The stages of ovule and anther development were analyzed by using a Leica DM5500B microscope with Nomarski DIC optics in 400x magnification (Leica Microsystems GmbH, Wetzlar, Germany). Images were taken by a DFC450C camera (Leica Microsystems GmbH, Wetzlar, Germany).