Alexa fluor 488 conjugated goat anti rabbit igg

After cortical neurons were cultured for 3 days, Aβ25–35 (10 μM) was simultaneously treated with PR extract (at doses of 0.1, 1, 10, and 100 μg mL^-1) for 4 days. After that, neurons were fixed with 4% PFA in PBS and blocked with 5% BSA in a solution of 0.3% Triton X-100 and PBS for 1 h at room temperature. The neurons were then incubated in a solution containing anti-pNF-H monoclonal antibody (dilution 1:500), anti-microtubule associated protein 2 polyclonal antibody (a neuronal marker; dilution 1:1000; Cat. No. ab32454; Abcam, Cambridge, United Kingdom), and 5% BSA in a solution of 0.3% Triton X-100 and PBS at 4°C overnight. Next, the neurons were incubated in a solution containing Alexa Fluor 594-conjugated goat anti-mouse IgG (dilution 1:400), Alexa Fluor 488-conjugated goat anti-rabbit IgG (dilution 1:400), DAPI (1 μg mL^-1, Sigma), and 3% BSA and 1% NGS in a solution of 0.3% Triton X-100 and PBS for 2 h at room temperature. Fluorescence images (650 μm × 852 μm) were captured using a 10× NA 0.4 dry objective lens on an inverted microscope (AxioObserver Z1). The lengths of axons per neuron were measured using MetaMorph 7.8 software (Molecular Devices, Sunnyvale, CA, United States).

Parasite conversion was investigated using coverslips with confluent CHO or HFF cells infected with the ME49 strain of T. gondii. The coverslips were collected 72 h after parasite inoculation, washed twice with PBS containing 1 mM CaCl₂ and 1 mM MgCl₂ (PBS++) and then fixed with 3% paraformaldehyde in PBS++. After washing twice with PBS++, the cells were permeabilized with 0.3% Triton X-100 in PBS++ for 5 min at room temperature. After washing, the coverslips were incubated with 3% bovine serum albumin (BSA) in PBS++ at room temperature for 30 min. The coverslips were incubated with BAG1 rabbit anti-serum and an anti-SAG1 mouse monoclonal antibody (clone TP3; Advanced ImmunoChemical Inc., Long Beach, CA) diluted at 1:100 in 3% BSA in PBS++ for 1 h at room temperature. After washing three times with PBS++, the coverslips were incubated with Alexa Fluor 488-conjugated goat anti-rabbit IgG and Alexa Fluor 594-conjugated goat anti-mouse IgG (Sigma, St. Louis, MO) diluted at 1:1000 in 3% BSA in PBS++ for 1 h at room temperature and then washed again with PBS++. The coverslips were placed on a glass slide coated with Mowiol (Calbiochem, San Diego, CA), and the slides were examined using a fluorescence microscope (Nikon, Tokyo, Japan). A total of 100 infected cells were counted and the percentage of BAG1-positive vacuoles was determined.

Cells were seeded in 8-well chamber slides (Thermo) and fixated with 3–4% formaldehyde for 15–30 min at room temperature. To detect HCV infection, cells were immunostained with human serum derived from HCV-infected patient, followed by incubation with anti-human Cy3 (Jackson) secondary antibody. To detect SARS-CoV-2 infection, the fixed cells were blocked with PBS containing 2% FBS and stained with hyperimmune rabbit serum from intravenous SARS-CoV-2-infected rabbits (in-house preparation), and then with Alexa Fluor 488-conjugated goat anti-rabbit IgG (Sigma). To detect ACE2 expression, cells were immunostained with mouse anti-ACE2 (Abcam) or goat anti-ACE2 (R &D systems), and then with anti-mouse 488 Alexa fluor IgG (Jackson) and anti-goat IgG (Invitrogen), respectively, and counterstained with DAPI. Images were obtained with a Zen Live Imaging (Time Lapse) microscope (Zeiss) or confocal microscope (LSM 780 + Chameleon Vision II).