Thunder imager model organism

The expression of MERS S, S1, and RBD proteins in mammalian cells was tested by infecting Huh7 cells with baculovirus at a multiplicity of infection (MOI) of 100. Three days after infection, immunofluorescence analyses and western blotting were carried out using a polyclonal antibody against MERS-CoV S protein (SICGEN, Portugal).
The expression of SARS-CoV2 S, S1, and MERS N-RBD proteins in mammalian cells was tested by infecting 293T cells with baculovirus at a MOI of 50. Three days after infection, immunofluorescence assays and western blotting were carried out using a polyclonal antibody against the spike RBD of SARS-CoV2 (Elabscience, USA).
The expression of the HERV gene in Sf9 cells following infection (3 MOI) with each baculovirus construct was assessed by western blotting using a polyclonal rabbit primary antibody specific for HERV Env (abcam, UK).
Immunofluorescence analyses were observed under a microscope (NIS-Elements-BR, Nikon or THUNDER Imager Model Organism, Leica). Western blots were developed using X-ray films. Uncropped figures are available in the Supplementary Information file. Marker bands have been indicated by their individual size. All western blots derive from the same experiment and were processed in parallel.

Live imaging was performed in ovo using a custom-made egg incubator designed for live observation as described in quailDB (http://quailnet.geneticsandbioinformatics.eu/). The transgenic quail eggs were carefully placed in a stainless-steel cup without damaging the egg yolk. Embryo turn to the top and the stainless-steel cup is filled up with egg white. A CultFoil 25 μm Teflon membrane (Zeiss, allowing gas exchange and avoiding dehydration) was placed over the embryo. The stainless- steel cup is then placed on a heat pad, which maintains the temperature of the embryo at 38°C. Embryos were imaged using a Leica Thunder Imager Model Organism (Videos 1 and 2) or under a Leica SP8 confocal upright (Videos 3 and 4).

Seven-days old seedlings were kept for 16 h at either 1%, 2.5%, or 21% v/v oxygen concentration and then used for GFP imaging. Imaging was performed with a Leica THUNDER imager model organism using bandpass filters for GFP (excitation: 470/40 nm, emission: 525/50 nm) and RFP (excitation: 546/10 nm, emission: 605/70 nm). Confocal laser scanning microscopy was performed using a Zeiss airyscan 800. Fiji was used to quantify UnaG and mCherry fluorescence intensity [38 (link)]. Each data point represents the average mCherry/UnaG ratio at the nuclei and cytosol.