A1 spectral detector confocal with flim module

Morphology of the β cell cluster within the primary Islet of Langerhaans was quantified in Tg(insulin:GFP) larval and juvenile zebrafish using confocal microscopy. At the conclusion of the exposure period on 15 or 30 dpf (detailed in section 2.3.2), zebrafish were gently washed in fresh 0.3X Danieau’s, anaesthetized, and low-magnification (20X) images were taken to measure fish length. Once anaesthetized and measured, fish were preserved in 4% paraformaldehyde. Preserved fish were stored in VECTASHIELD® Antifade Mounting Medium (Vector Laboratories, Burlingame, CA) until imaging. Zebrafish were staged on glass slides and imaged laterally using a Nikon A1 Spectral Detector Confocal with FLIM Module at the University of Massachusetts Light Microscopy Core Facility located within the Institute for Applied Life Sciences. To obtain a 3-dimensional islet structure, z-stacks were obtained by taking photographs at 1.8 μm steps. β cell volumes and sphericity were calculated using the Nikon NIS Elements Advanced Research software package. Larvae were obtained from 3–4 experimental replicates with 19–43 larvae per group at 15 dpf and 22–39 fish per group at 30 dpf.

Immunofluorescence staining (IF) was carried out in accordance with the methods of our previous studies (Miao et al. 2020 (link)). E3.0 morula embryos were harvested and then fixed directly in 4% paraformaldehyde. After brief wash in PBS, embryos were permeabilized with 0.5% Triton X-100 for 20 min. Embryos were then blocked in blocking solution (PBS with 10% FBS and 0.1% Triton) for 1 h at room temperature, and then incubated with primary antibodies overnight at 4 °C. All primary antibodies were diluted 1:200 using the blocking solution, including: goat anti-OCT4 (abcam, ab27985); mouse anti-CDX2 (BioGenex, MU392A-UC); rabbit anti-TRP53 (Cell Signaling Technology, #9284). After 3 washes, embryos were incubated with suitable secondary antibodies (Alexa Fluor, Thermo Fisher Scientific, Waltham, MA, USA) for 1 h at room temperature in dark. After 2 washes, DNA was stained with 4,6-diamidino-2-phenylindole (DAPI) before transferring embryos into single wells of chambered slides (Corning Co., Corning, NY, USA) for imaging under a Nikon A1 Spectral Detector Confocal with FLIM Module. Z-stacks (20X objective, 8 μm sections) were collected and maximum projection applied. Embryos were handled individually such that each one was imaged and then recovered for PCR genotyping.

Arabidopsis roots stably expressing 35Sp-FPN3-GFP were imaged with the Nikon A1 Spectral Detector Confocal with FLIM Module at the Light Microscopy Core, Institute for Applied Life Science at University of Massachusetts, Amherst. For mitochondrial co-localization roots were stained with MitoTracker Red FM (Invitrogen) to label the mitochondria prior to imaging, by incubating roots in a final concentration of 1 μM MitoTracker Red FM for 30 minutes. Z-stack images were taken with FITC and TRITC channels. Images were collected and processed with NIS-Elements software.