Gtx632604

IFA was conducted for the detection of SARS-CoV-2 spike proteins. At 24 hpi, the supernatants were removed, and the cells were fixed using 4% paraformaldehyde in PBS for 10 min at 25 °C. The fixed cells were washed three times with ice-cold PBS. To detect the intracellular viral proteins, the cells were permeabilized by treating with PBS containing 0.5% Triton X-100 for 10 min. After permeabilization, the cells were washed in PBS three times for 5 min in each round. The cells were then blocked with 1% bovine serum albumin (BSA) in PBS-T for 30 min at 25 °C. Next, an anti-SARS-CoV-2 spike protein antibody (GTX632604, GeneTex) was diluted in PBS-T with 1% BSA, and the blocked cells were treated using this as the primary antibody for 1 h in a humidified chamber at 25 °C. After the primary antibody reaction, the solution was decanted, and the cells were washed three times in PBS for 5 min in each washing step. For the secondary antibody reaction, the cells were treated with Alexa Fluor 488-conjugated anti-mouse IgG antibody in PBS-T with 1% BSA for 1 h at 25 °C in the dark. Following this, the solution was decanted, and the cells were washed three times in PBS for 5 min in each washing step. Counter staining was performed using 4′,6-diamidino-2-phenylindol.

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).

Automated IHC on 4 µm-thick FFPE brain and lung sections using SARS-CoV/SARS-CoV-2 Nucleocapsid (Sino Biological, 40,143-R019, clone 019, dilution 1:10,000) and Spike (GeneTex, GTX632604, clone 1A9, dilution 1:100) antibodies were processed on Dako Omnis (Agilent Technologies, Santa Clara, CA, USA). Negative tissue controls were obtained from patients who had an autopsy before the COVID-19 pandemic. IHC evaluation was performed by three pathologists (LL, SD, IS) as follows: positive (+) and negative (−), i.e., no staining or scattered positive cells. Additional information is available in Additional file 4.