240 c igg2a

Briefly, 293 LTV cells transfected with pIDV-V1 and pIDV-V5 plasmids encoding SARS-CoV-2 spike glycoprotein and Spike fused with 5mer4 respectively. Cells were lysed at 48 h post-transfection by xTractor™ Buffer (Takara Bio USA, Inc.) according to the manufacturer’s instructions. Following this, lysates were centrifuged at 12,000 × g for 20 min at 4 °C. Next 5 µg of cleaned samples were separated in a Bis-Tris, 1.0 mm, Mini Protein Gel (Life Technologies, Canada), and transferred onto nitrocellulose membranes using the iBlot 2 Gel Transfer Device (Life Technologies, Canada). The membrane was blocked overnight at 4 °C with PBS containing 5% milk and 0.1% Tween 20 (Bio-Rad, Canada) and then blotted with a mixture of mouse Anti-SARS-CoV S Protein 154 C IgM (BEI Resources, USA), 240 C IgG2a (BEI Resources, Manassas, USA), 540 C IgG2a (BEI Resources, Manassas, USA), and 341 C IgG2a (BEI Resources, Manassas, USA) (Supplementary Fig. 1). A goat anti-mouse human peroxidase-conjugated labeled antibody (Invitrogen, Canada) was used as the secondary antibody, followed by visualization using the Clarity ECL Western Blotting Detection Substrate (Bio-Rad, Canada). The unprocessed gel is shown in Supplementary Fig. 3.

Mice were immunized with 100 µg of pIDV-II-SARS-CoV-2 spike or TE buffer (control group) using IONAID and then euthanized at 24 h, 48 h, or 72 h post-immunization. Mouse-skin specimens were dissected at the tattooing site and were fixed in 10% neutral phosphate-buffered formalin, routinely processed, and sectioned at 5 μm. Samples were subjected to heat-mediated antigen retrieval with citrate buffer and stained in a 1:1:1:1 ratio for SARS-CoV-2 S protein using the following antibodies, each diluted 1:1000: mouse anti-SARS-CoV S Protein 154C IgM (BEI Resources, Cat. NR-620), 240C IgG2a (BEI Resources, Cat. NR-616), 540C IgG2a (BEI Resources, Cat.NR-618), and 341C IgG2 (BEI Resources, Cat. NR-617) followed by incubation with Mach3 mouse probe (BioCare Medical) and DAB Substrate (BioCare Medical), respectively. Then, the slides were counter-stained with hematoxylin prior to dehydration with 95% and 100% ethanol. Images were acquired on a Huron Digital Pathology Tissue Scope LE slide scanner under the ×10 objective.