Vectashield antifade mounting medium with dapi

Cells were cultured in 8 chamber slides (Falcon™ Chambered Cell Culture Slides, Fisher Scientific, Canada) until reaching confluency of 80–90% before staining. Each biological sample was cultured and stained in doublets. BrdU (Cell Signaling Technology Inc., Canada) was added to the culture medium (1:200) and incubated with the cells for 12 h prior. The cultured cells were fixed in 4% paraformaldehyde (Electron Microscopy Sciences, USA) followed by permeabilization with 0.25% Triton X-100 (BioShop Canada Inc., Canada) and incubated in 1.5 M hydrochloric acid (Sigma Aldrich, Canada). To prevent unspecific binding, PBS was added with 1% bovine serum albumin (WISENT Inc., Canada). The samples were incubated with the primary antibody (BrdU (Bu20a) Mouse mAb #5292, Cell Signaling Technology Inc., Canada) at 4 °C overnight, followed by a 1-h long incubation at room temperature in the secondary antibody (Alexa Fluor® 488 dye, Thermo Fischer Scientific, Canada). Afterward, the slides were mounted with mounting medium containing DAPI (VECTASHIELD Antifade Mounting Medium with DAPI, Vector Laboratories, USA).

Antibodies and reagents used for immunostaining are listed here: Anti-acetylated α-tubulin (Cell Signaling, cat#5335S), anti-DPP4 (R&D Systems, cat#AF1180), anti-MERS-CoV N protein (Sino Biological, cat#40068-MM10), anti-cleaved caspase-3 (Cell Signaling, cat#9664), and Alexa Fluor 647 phalloidin (ThermoFisher Scientific, cat#A22287). Methods for immunolocalization of DPP4 in human airway epithelia were adapted from previous studies (7 (link)). Briefly, airway epithelia or cytospun BAL samples were fixed in 4% paraformaldehyde and permeabilized with 0.2 % Triton X-100. Following incubation with SuperBlock™ buffer, primary antibodies were applied to the fixed cells on slides, or to the apical and basolateral cell surfaces of epithelial sheets overnight, followed by washing with SuperBlock™ buffer three times. After a 1 h incubation with the appropriate Alexa Fluor secondary antibodies, followed by more washes, coverslips were mounted using Vectashield antifade mounting medium with DAPI (Vector Laboratories). Photomicrographs were taken using a Leica TCS SPE confocal microscope. The number and types of infected cells were counted manually using ImageJ.

Synchronous populations of worms were grown on NGM plates until adulthood and were carefully monitored using a stereomicroscope and bleached shortly after worms started to produce the first embryos. After bleaching, Early embryos were washed with cold M9 buffer to slow down embryonic development and immediately frozen in dry ice. A small aliquot of embryo pellet (2 µL) was taken right before freezing and mixed with 10 µL VECTASHIELD® Antifade Mounting Medium with DAPI (Vector laboratories) and immediately frozen in dry ice. DAPI-stained embryos were defrosted on ice and used for counting cell nuclei and score the embryonic cell stage of the population.

T. thermophila wild type and partial MLP1 knockout strains were grown to mid-log phase and prepared for indirect immunofluorescent staining as described^{62 (link)}. Centrifugation steps, including washes, were performed for 3 min at 1000 × g. Briefly, fixative (two parts saturated HgCl₂ and one part 95% ethanol) was added to the cell suspension and incubated for 5 min at room temperature, followed by collecting and resuspending the cell pellet in 100% ice-cold methanol twice. The cell pellet was washed with PBS and incubated with primary rabbit anti-Mlp1 antibody (1:500 dilution) rotating overnight at 4 °C. The cell pellet was washed three times with PBS, followed by incubation with secondary goat anti-rabbit IgG (1:10,000, ThermoFisher Scientific A11008), rotating for 1 h at room temperature. The cell pellet was washed three times with PBS. The cell suspension was dropped on a coverslip, air-dried, and mounted with Vectashield Antifade Mounting Medium with DAPI (Vector Laboratories) onto a microscope slide. Microscopy images were obtained at 63x magnification on a LSM700 confocal laser scanning microscope (Zeiss) and processed in ZEN3.3 (blue edition).