Sp8 x inverted confocal microscope

The full length coding sequence of Gs5PTase8 without the stop codon was amplified and cloned into the XbaI and BamHI restriction enzyme sites of the binary vector pTEV8 at the downstream of 35S promoter. The GFP coding sequence was cloned behind Gs5PTase8 between the sites of BamHI and XhoI. The constructs were coated with gold particles and bombarded into the epidermal cells of onion (Bio-Rad PDS-1000/He system, Hercules, CA, USA). The onion sections were imaged at room temperature using a Leica SP8 X inverted confocal microscope with an Argon laser (Leica, Wetzlar, Germany). Green fluorescence protein (GFP) is excited at 488 nm and the emitted light is captured at 510-525nm. The images were captured digitally and processed using the Leica Application Suite Advanced Fluorescence Lite (LAS AF version: 2.6.3 build 8173). Plasmolysis was performed by incubating the samples in 25% sucrose for 5 min.

Cells were seeded in 24-well plates containing coverslips. Plates were infected or transfected depending on the assay. Cells were fixed using 4% paraformaldehyde for 30 min at room temperature, then permeabilized in PBS buffer containing 3% bovine albumin serum (BSA) and 0.1% Triton X-100 for 30 min at room temperature. Cells were then washed once in PBS-3% BSA-0.05% Tween20 and incubated with relevant mouse or rabbit primary antibody (anti-F 1:1000, anti-N 1:20,000; anti-GM130 1:1000) diluted in PBS-3% BSA-0.05% Tween20 for 1 h at room temperature. Cells were washed 3 times with PBS-3% BSA-0.05% Tween20, then incubated with goat anti-mouse or goat anti-rabbit secondary antibody coupled to Alexa 488 or Alexa 594, respectively. Cells were then washed 3 times with PBS-3% BSA-0.05% Tween20 and two times with PBS. For nucleus labeling, cells were exposed to Hoechst 33,342 stain (Invitrogen) during incubation with secondary antibodies. Coverslips were mounted with ProLong Gold antifade reagent (Invitrogen). Images were obtained using an inverted SP8x confocal microscope (Leica, Welzlar, Germany) using a 63× oil immersion objective. Maximum projections of optical Z slices are shown. Images were processed with Leica imaging software LAS X 1.4.5 and ImageJFIJI software version 2.3.0.

To generate 4D datasets, 4.5 hpf old embryos were mounted on glass bottom microwell petri dishes (MatTek, part# P35G-1.5–20-C) in artificial seawater. Plates were sealed by piping a border of a mix of Vaseline and 5% (v/v) mineral oil (Sigma, item #M841–100 ml) and covered with a 22 × 22 Fisherbrand Cover Glass (item # 12–541-B). Embryos were imaged on a Leica inverted SP8 X Confocal microscope every 3.5 min for 4–5 h. B7.5 lineage nuclei and epidermal cell membranes were visualized using Mesp > H2B::GFP and EfnB > hCD4::mCherry^{13 (link)}, respectively, and TVC migration was tracked using Bitplane Imaris Software Spots module.
To express TVC position during migration in 3D we subdivide the embryo into quadrants using two conceptual orthogonal planes that bisect the developing embryo. The position of the sagittal plane is determined by the midline of the epidermis visible using the EfnB > hCD4::mCherry transgene. The frontal plane is orthogonal to the mid-sagittal plane and passes through the nucleus of the anterior ATM and the future position of the palps, the most anterior point of the embryo. Imaris (Bitplane) is used to calculate a vector line based on position of GFP + TVC nuclei.

To generate 4D datasets, embryos at 4.5 hpf FABA stage 15 were mounted on glass-bottom microwell Petri dishes (MatTek, part# P35G-1.5–20C) in artificial seawater. Plates were sealed by piping a border of vaseline and 5% (v/v) mineral oil (Sigma, #M841-100ml) and covered with a 22 × 22 Fisherbrand Cover Glass (#12-541-B). Embryos were imaged on a Leica inverted SP8 X Confocal microscope using the 40× water immersion lens at 512 × 512 resolution every 3.5 min for 4–5 hr. B7.5 lineage nuclei and epidermal cell membranes were visualized using Mesp >H2B::GFP and EfnB>hCD4::mCherry, respectively, and TVC migration was tracked using Bitplane Imaris Software Spots module.