Goat anti human alexa fluor 488

Cells were cultured
in 6-well plates to confluency. To induce CAIX expression, cells were
cultured under hypoxic conditions of 1% O₂ for 24, 48,
or 72 h in a Whitley H35 Workstation (Don Whitley Scientific). The
binding of radiolabeled antibodies and 50% inhibitory concentration
(IC₅₀) were determined as described by Heskamp et al.^{31 (link)} The percentage of CAIX⁺ cells was
determined by flow cytometry analysis on a FACSCanto II (BD Bioscience).
Cells were harvested, and fixable viability dye eFluor 780 (eBioscience,
65-0865-14) was added to distinguish dead and live cells. Cells were
stained with Human IgG1 anti-mouse CAIX (MSC3) for 30 min at 4 °C
and with goat anti-human Alexa Fluor 488 (Invitrogen, A-11013) for
15 min at 4 °C. Data were analyzed using FlowJo V10.7 (Tree Star).

For the IFA, BHK cells transfected with pCAG-SARS-CoV-2 S-full were fixed with acetone-methanol (1:1) at 4 °C for 20 min. Fixed cells were reacted with the test serum samples, which were diluted at 1:100 with PBS. After an incubation for 1 h, the cells were rinsed with PBS and incubated with goat anti-human Alexa Fluor 488 (Invitrogen). After washing with PBS, staining was observed under a fluorescence microscope.

Indirect immunofluorescence was performed as previously reported (32 (link)). In brief, 36 h after transfection, coverslips were washed three times with PBS, fixed with 4% (w/v) paraformaldehyde in PBS for 10 min at room temperature, rinsed three times with PBS, and permeabilized with 0.1% (v/v) Triton-X100 (Sigma) in PBS for 15 min at room temperature. Next, 1 ml 1% BSA/PBS was added to the coverslips for 30 min at room temperature to block unspecific binding of antibodies. Primary antibodies (rabbit anti-SARS-CoV-2 spike protein monoclonal antibody, 40150-R007, Sino Biological, 1/100 dilution; human anti-SARS-CoV-2 4A8 monoclonal antibody, CPC514A, Cell Sciences, 1/100 dilution in PBS with 1% BSA) were added and incubated at 4°C overnight. Coverslips were washed with PBS three times, and secondary antibodies (Goat anti-Rabbit Alexa Fluor Plus 555 Invitrogen, and Goat anti-Human Alexa Fluor 488, Invitrogen, 1/400 dilution in PBS with 1% BSA) were added and incubated at room temperature for 1 h. Coverslips were washed with PBS three times and counterstained with DAPI (Invitrogen). Immunofluorescence images were acquired with a Leica SP8 X Confocal Microscope (Lecia) using LAS X software.

Animals were anesthetized with a lethal dose of beuthanasia-D solution and transcardially perfused with 4% paraformaldehyde. Whole brains were removed, processed, embedded in paraffin, and cut into 4–6 μm sections. After de-parafinization, slides were incubated for 10 min at 95°C in Trilogy solution (Cell Marque), blocked with 3% hydrogen peroxide, followed by 2% normal goat serum. The sections were incubated with TBI or normal human serum overnight at 4°C, then incubated with goat anti-human HRP (Abcam) diluted in 2% goat serum. Staining was visualized with 3,3′-diaminobenzidine (DAB; Dako). The sections were counterstained with hematoxylin (Dako). Sections were finally washed with PBS, mounted, air-dried and cover slipped with Aquamount (Dako). The slides were scanned and examined using the Aperio ScanScope GL system at 20 x and ScanScope software. For immunofluorescence experiments, sections were incubated with anti-GFAP Alexa Fluor 555 conjugate (Cell Signaling) and either normal or TBI human serum overnight at 4°C, followed by goat anti-human Alexa Fluor 488 (Life Technologies) diluted in 2% goat serum. The sections were washed with PBS, mounted, air-dried and cover slipped with FluoroGel (GenTex). Staining was imaged with a Zeiss fluorescence microscope.

Hela
S3 cells at 4 × 10⁵ cell/mL were infected with 10
MOI ChAdOx1 nCoV-19 and incubated at 37 °C for 40–48 h.
Cells were harvested and pelleted by centrifugation at 500g for 5 min. Resuspended cells were split into three pools
for the alternative staining and were incubated with either pooled
prime-boost sera derived from outbred CD1 mice immunized intramuscularly
with 10⁸ infectious units of ChAdOx1 nCoV-19,^{18 (link)} prepared at 1:50 in PBS-BSA-0.5% or human mAbs
prepared at 1 μg/mL or recombinant ACE2 expressed with a human
Fc tag prepared at 2 μg/mL (see below). Cells were incubated
for 2 h at rt. Cells were washed 3× with PBS-BSA-0.5% and then
incubated for 1 h at rt with secondary antibodies conjugated with
Alexafluor488 diluted at 1:1000 in PBS-BSA 0.5% (for ChAdOx1 nCov-19
serum staining Goat Anti mouse AlexaFluor 488 (Life Tech A11029),
for ACE2 staining, Goat Anti Human AlexaFluor 488 (Life Tech)). Cells
were washed twice with PBS-BSA-0.5% before resuspending in PBS and
analyzed by flow cytometry using a Fortessa X20 FACS analyzer. Samples
were considered positive for spike expression if they had a fluorescence
intensity above a threshold value determined by the maximum intensity
of the noninfected control cells (Figure 1D). Experiments were performed twice in duplicate,
and representative data are shown. Data were analyzed using FlowJo
v9 (TreeStar).