Immunostaining and TUNEL Assay for SARS-CoV-2

Mock and infected LC, SC, or STC grown on glass coverslips were fixed with 4% PFA, permeabilized with 0.1% Triton X-100 in PBS, and blocked with 5% bovine serum albumin in PBS. Cells were then incubated with primary antibodies against anti-Spike (GeneTex, GTX632604 at 1:500 dilution), followed by fluorophore-conjugated secondary antibody (Invitrogen Alexa Fluor 488-conjugated sheep anti-rabbit, 1:5000 dilution), and examined using an Axiocam MR camera mounted on a Zeiss Axiovert 200 microscope. TUNEL assay was performed using the Promega Fluorometric TUNEL System according to the manufacturer’s instructions. Undifferentiated spermatogonia were also stained for the well-established cell-specific marker, ubiquitin carboxyl-terminal esterase L1 (UCHL1) [31 (link)] using rabbit anti-human UCHL1 (Sigma, HPA005993 at 1:1,000 dilution). The secondary antibody was Alexa Fluor 594-conjugated goat anti-rabbit (Invitrogen; 1:5000 dilution).