Biotinylated anti mouse igg antibody

Mice were anesthetized and perfusion-fixed with 4% paraformaldehyde (PFA) after treatments with ASC-CM or BME. PFA-fixed and paraffin embedded spinal cords were sectioned (15 μm) serially via the lumber enlargement and immunostained with anti-microtubule-associated protein 2 antibody (1:1000, MAP2; Millipore), then followed by a biotinylated anti-mouse IgG antibody (Sigma-Aldrich Corp.). Sections were visualized under the microscope after applying 3,3′-diaminobenzidine (DAB) substrate solution. A 40x objective light microscope (Nikon, Japan) was used to count large motor neurons in the ventral horn of the lumbar spinal cord39 (link)40 (link). 6–8 sections of each lumbar were counted. Data was reported as number of motor neurons per section as previously described33 (link)41 (link).

The plaque assay was performed as described previously [31 (link)]. Briefly, confluent ST cells on a 12-well plate were inoculated with 0.1 mL each of 10-fold serially diluted viruses in MEM/BSA and incubated for 1 h at 37 °C. Cell were then washed with MEM/BSA, covered with 1 mL of MEM/BSA containing TPCK-trypsin (0.5 μg/mL) and 1% Seakem GTG agarose (Lonza Japan, Chiba, Japan), and incubated at 37 °C for 3 days. Subsequently, 30% formalin in PBS (0.5 mL) was added to each well to fix the cells at 4 °C overnight. After formalin and agarose were removed, the cells were washed with PBS and permeabilized with 0.1% Triton X-100 in PBS for 15 min at 23 °C. After blocking with BlockAce (KAC, Hyogo, Japan), the cells were incubated with anti-D/OK mouse immune serum for 60 min, biotinylated anti-mouse IgG antibody (#B7264, Sigma-Aldrich Japan, Tokyo, Japan) for 30 min, and then a complex with streptavidin (8 µg/mL) (Fujifilm Wako Chemicals, Miyazaki, Japan) and biotinylated peroxidase (4 µg/mL) (Invitrogen/Thermo Fisher Scientific, Tokyo, Japan) for 30 min. The plaques were visualized using a DAB peroxidase substrate kit (Vector Laboratories, Burlingame, CA, USA), according to the manufacturer’s instructions.

The plaque assay was performed as described previously^{38 (link)}. Briefly, confluent ST cells on a 12-well plate were inoculated with 0.1 mL of each virus serially diluted in MEM/BSA and incubated for one hour at 33 °C or 37 °C. Cells were then washed with MEM/BSA, covered with 1 mL of MEM/BSA containing TPCK-trypsin (0.5 µg/mL) and 1% Seakem GTG agarose (Lonza Japan, Chiba, Japan), and incubated at 33 °C or 37 °C for two–three days. Subsequently, 30% formalin in PBS (0.5 mL) was added to each well to fix the cells at 4 °C overnight. After formalin and agarose were removed, the cells were washed with PBS and permeabilized with 0.1% Triton X-100 in PBS for 15 min at 23 °C. After blocking with BlockAce (KAC, Hyogo, Japan), the cells were incubated with anti-IDV mouse immune serum for 60 min, biotinylated anti-mouse IgG antibody (#B7264, Sigma) for 30 min, and a complex comprising streptavidin (8 µg/mL) (Fujifilm Wako Chemicals, Miyazaki, Japan) and biotinylated peroxidase (4 µg/mL) (Invitrogen/Thermo Fisher Scientific, Tokyo, Japan) for 30 min. The plaques were visualized using a DAB peroxidase substrate kit (Vector Laboratories, Burlingame, CA, USA) according to the manufacturer’s instructions.