Goat anti mouse antibody conjugated to alexa fluor 488

Mice were deeply anesthetized with 0.14 g/kg sodium pentobarbital, intracardially perfused with PBS, and fixed using 4% paraformaldehyde. Serial coronal sections (35 μm) were cut with a sliding microtome (Leica Biosystems, Buffalo Grove, IL, USA) and stained as freely floating sections. For examination of amyloid plaques, sections were incubated in 88% formic acid at room temperature for 8 minutes. After a washing step in PBS at pH 7.4, sections were blocked in PBS containing 5% bovine serum albumin and 0.5% Triton X-100 for 2 h at 37 °C, then incubated with mouse anti-Aβ antibody (1:1000, Covance 6E10; BioLegend, San Diego, CA, USA) overnight at 4 °C. After further PBS washing, sections were incubated with goat antimouse antibody conjugated to Alexa Fluor 488 (1:500; Thermo Fisher Scientific, Waltham, MA, USA) for 2 h at 37 °C and mounted on (3-aminopropyl)triethoxysilane-coated glass slides. Z-stack images were taken using a Zeiss Pascal (Carl Zeiss Microscopy, Jena, Germany) or Nikon A1 (Nikon Instruments, Tokyo, Japan) laser scanning microscope with a ×20 lens objective. Results were quantified using Image-Pro Plus (Media Cybernetics, Rockville, MD, USA).

Hepa 1.6 cell were seeded onto 2 cm² coverslips (Millipore Sigma) and transfected as described above. After 48 h, cells were fixed with 4% v/v paraformaldehyde, permeabilized with 0.1% v/v Triton X-100, and immunostained with anti-human AAT (Dako, Agilent, Stockport, UK) (2.2 μg/mL) or the anti-AAT polymer 2C1 mAb (0.8 μg/mL) overnight at 4 °C. The primary antibodies were respectively detected with goat anti-mouse antibody conjugated to Alexa Fluor 488 and goat anti-rabbit antibody conjugated to Alexa Fluor 555 (Thermo Fisher Scientific, Loughborough, UK), respectively. Cells were counterstained with Hoechst (Thermo Fisher Scientific, Loughborough, UK) to visualize the nuclei. Coverslips were mounted on slides with Immuno-Mount (Thermo Fisher Scientific, Loughborough, UK) and analyzed on a Zeiss LSM700 confocal microscope with a 63× objective (1.4 oil).

In order to titrate the UK661 and F52/70 viruses, 96-well U-bottomed plates (Thermo Fisher Scientific) were seeded with the immortal B cell line, DT40, at a seeding density of 1 × 10⁴ cells per well in 180 μL media. A 10-fold dilution series of the UK661 or F52/70 viruses was added to the cells, with 20 μL of each dilution added to each well in quadruplicate. Cells were incubated at 37°C for 5 days, fixed in 4% paraformaldehyde and stained with a primary mouse monoclonal antibody raised against IBDV VP2 (Wark, 2000 ) and a secondary goat anti-mouse antibody conjugated to Alexa Fluor 488 (Thermo Fisher Scientific). Wells were marked positive or negative for the presence or absence of virus by immunofluorescence microscopy and the TCID₅₀/mL calculated by the Reed and Muench method (Reed and Muench, 1938 (link)). In order to titrate the PBG98 and chimeric viruses, 96-well plates were seeded with DF-1 cells at a density of 1 × 10⁴ cells per well in 180 μL media. A 10-fold dilution series of the PBG98 or chimeric viruses were added to the cells, with 20 μL of each dilution added to each well in quadruplicate. Cells were incubated at 37°C for 5 days, and wells were marked positive or negative for the presence or absence of cpe and the TCID₅₀/mL calculated by the Reed and Muench method (Reed and Muench, 1938 (link)).