Leica mzfliii microscope

Seeds at 15 days after flowering (DAF) were collected and fixed overnight in 4% paraformaldehyde (Sigma, Santa Clara, CA, USA), then dehydrated in an ethanol gradient series (30, 50, 70, 85, 95, and 100% ethanol), and embedded in Paraplast Plus (Sigma, Santa Clara, CA, USA). The sample blocks were sectioned into 8-μm slices using a Leica RM2265 microtome (Leica Microsystems, Wetzlar, Hesse-Darmstadt, Germany) and stained with 0.5% toluidine blue. Images were captured using a Leica MZFLIII microscope (Leica Microsystems, Wetzlar, Hesse-Darmstadt, Germany).

Kernels at 6, 12, and 21 DAP were harvested from the selfed +/Zmsmk3 heterozygotes and fixed overnight in 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA), dehydrated in an ethanol gradient series (30%, 50%, 70%, 85%, 95%, and 100% ethanol), and embedded in Paraplast Plus (Sigma-Aldrich). The samples were sectioned into 8–12 µm slices using a Leica RM2265 microtome (Leica Microsystems, Wetzlar, Germany) and stained with 0.5% toluidine blue O. Images were captured using a Leica MZFLIII microscope (Leica Microsystems).
For the TEM analysis, the 10-DAP endosperms of the WT and Zmsmk3 kernels were fixed, washed, dehydrated, embedded, and cut into ultrathin sections, as previously described (Ren et al. 2017 (link)). Ultra-thin sections were obtained using a Leica EM UC7 ultra microtome (Leica Microsystems). The sections were stained with uranyl acetate and subsequently with lead citrate, then imaged using a Tecnai G² 20 TWIN transmission electron microscope. These procedures were performed by Pei Zhang (Core Facility and Technical Support, Wuhan Institute of Virology, China).

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.

Whole-mount images were taken using a Leica MZFLIII microscope (Leica Microsystems, Wetzlar, Germany) fitted with a QImaging MicroPublisher 5.0 RTV camera and QCapture Pro 6.0 software (QImaging, Surrey, BC, Canada); a Zeiss AxioSkop2 microscope fitted with a Zeiss AxioCam HRc camera and Zeiss AxioVision Rel. 4.8 software (Carl Zeiss, Oberkochen, Germany); an Olympus MVX10 microscope (Olympus Corporation, Tokyo, Japan) fitted with a Zeiss AxioCam HRc camera and Zeiss AxioVision Rel. 4.8 software; an Olympus 1 × 71 inverted microscope fitted with a Hamamatsu ORCA-R2 monochrome camera and HCImage software (Hamamatsu Photonics, Hamamatsu, Japan); a Zeiss LSM 710 confocal microscope with Zeiss ZEN software; and a Zeiss 710 confocal microscope with Zeiss LSM Image Browser (version 4.2.0.121) software, which was used to create three-dimensional images and stack movies. Images of sections were taken using a Zeiss AxioSkop 2 MOT microscope fitted with a QImaging Retiga 2000R camera, a Qimaging RGB pancake and QCapture Pro 6.0 software; a Zeiss Scope.A1 microscope fitted with a Zeiss AxioCam MRm camera and Zeiss ZEN 2012 (blue edition) software; and a Zeiss LSM 780 confocal microscope with Zeiss ZEN 2011 (black edition) software. All images were further processed in Photoshop CS4 (Adobe Systems Inc., San Jose, CA) and/or ImageJ 1.50i software (NIH, Bethesda, MD).