Sp8 confocal multiphoton microscope

Embryos were live imaged using a Leica SP8 Multi-photon confocal microscope. The wavelengths for confocal excitation were 514 nm laser (for mVenus of R26Fucci2a), 488 nm (for CerI-GFP) while for multi-photon excitation 1040 nm was used for R26Fucci2a embryos (when double-transgenic embryos R26Fucci2a/CerI-GFP embryos imaged). Fixed embryos were imaged on either Leica SP5 or SP8 confocal microscopes.

Cell viability was observed using a live/dead double staining assay (R37601, LIVE/DEAD^® Cell Imaging Kit, Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s instructions. Briefly, the scaffolds were washed twice with PBS and then fully submerged in the assay dye. After incubating in the dark for 15 min at room temperature, the scaffolds were sectioned with a sterile scalpel to expose the cross-sectional area which was viewed under a Leica SP8 Multiphoton Confocal microscope. Images were taken from the middle of the scaffold. Image J v1.51q (National Institute of Mental Health, Bethesda, MD, USA) was utilised to calculate the percentage of cell viability in the scaffolds as the percentage of living cells among the total number of cells visualised.

Single-labeled (c-Fos) cells, double-labeled (c-Fos + ChAT or PARV) cells, and AChE background staining were visualized using a Nikon E600 microscope fitted with a CoolSNAP digital camera (Roper Scientific, Trenton, NJ, United States). Fluorescence images were visualized using a Nikon E600 microscope or a Leica SP8 multiphoton confocal microscope (Leica Microsystems, Wetzlar, Germany) equipped with LAS AF 3 analysis software (Leica Microsystems, Wetzlar, Germany). Immunoperoxidase photomicrographs were captured using IP Lab Software (Scanalytics, Trenton, NJ, United States). Images were imported into Adobe Photoshop 6.0 (Adobe Systems, San Jose, CA, United States) to adjust the image size and to make minor alterations to contrast and brightness. Brain regions where photomicrographs were captured are highlighted in the results via modified illustrations from the Paxinos and Watson (1998) rat brain atlas.

Transverse sections of all grass leaves were imaged for light microscopy under 10× and 40× objectives using a Nikon Eclipse 50i upright microscope (Nikon Instruments). SEM was performed using a Zeiss Ultra Plus field emission scanning electron microscope at 3 kV. To quantify pit field distribution, two z-stacks from two leaf tissues per species were obtained using a Leica SP8 multiphoton confocal microscope (Leica Microsystems). Details can be found in Danila et al. (2016) (link).

Tissue from the widest portion of the third fully expanded leaf of 10-day-old plant was fixed with 4% paraformaldehyde for 1 h at room temperature, sectioned, and stained along with transverse sections of the root^{37 (link)}. Aliphatic suberin staining was performed using 0.05% Nile Red in ClearSee solution (10% (w/v) xylitol, 15% (w/v) sodium deoxycholate, 25% (w/v) urea in water) while lignin staining used 0.2% Basic Fuchsin in ClearSee solution; 0.1% Calcofluor White was used in combination with Nile Red and Basic Fuchsin to visualise cell walls. Nile Red-stained and Basic Fuchsin-stained tissues and sections were mounted onto glass slides with ClearSee solution. Fluorol Yellow staining was performed according to Yadav et al.³¹ . Slides were examined with a Leica SP8 multiphoton confocal microscope (Leica Microsystems). Nile Red was imaged at 561 nm excitation wavelength and detected at 600–620 nm. Basic Fuchsin was imaged at 561 nm excitation wavelength and detected at 600–650 nm. Fluorescence from Calcofluor White-stained cell walls was detected at 434–445 nm following excitation at 405 nm. Fluorol Yellow was imaged at 514 nm excitation wavelength and detected at 516–593 nm.