Alexa fluor 555 donkey anti mouse igg h l

E12.5 mouse lungs were fixated for 1 hr in 4% paraformaldehyde in PBS, and subsequently incubated with Lamin B1 (Thermo; Material No. 702972; 1:200) at 4°C for 3 days. As a structural component of the nuclear lamina, LaminB1 immunostaining makes crowded nuclei clearly distinguishable and easily segmentable. After washing in D-PBS, lungs were incubated with conjugated fluorescent secondary Alexa Fluor 555 donkey anti-mouse IgG (H+L) (Abcam; Material No. ab150106; 1:250) for 2 days at 4°C.

Cells on slides were fixed with 4% paraformaldehyde (PFA) for 20 min at room temperature and were permeabilized with 0.1% Triton X-100 in 1 × PBS for 5 min and blocked with blocking buffer (1% BSA and 2% donkey serum diluted in PBS) for 30 min. Immunofluorescence analyses of SARS-CoV-2-infected cells were performed using a rabbit anti-SARS-CoV-2 spike S/S2 protein antibody (1:500, 40590-T62, S&B), a mouse anti-dsRNA antibody (1:200, 10010200, J2-1909, SCICONS), an anti-ACE2 antibody (1:100, A12737">A12737, Abclonal), IRF-3 (D6I4C) XP rabbit mAb (1:300, #11904, CST), Alexa Fluor 680 donkey anti-rabbit IgG (H+L) (1:1000, ab175772, Abcam), and Alexa Fluor 555 donkey anti-mouse IgG (H+L) (1:1000, ab150106, Abcam). All cells were mounted with ProLongTM Gold Antifade with DAPI (Life Technologies, P36931) and imaged with a TissueFAXS 200 flow-type tissue cytometer (TissueGnostics GmbH, Vienna, Austria). All statistical analyses of immunofluorescence staining present the results from at least 3000 cells per replicate, and data are shown as the mean ± s.e.m.