Horseradish peroxidase conjugated anti mouse antibody

96-well ELISA plates (Corning Life Sciences, Corning, NY) were coated with 100 ng peptide in 100 µL PBS per well using the peptide used for immunization (see Table 1). Wells were washed with PBS and blocked with PBS/5% fetal bovine serum (FBS). Primary antibodies were added to blocking solution and incubated at room temperature. After PBS washes, plates were incubated with horseradish peroxidase-conjugated anti-mouse antibody (Jackson Immuno Research Labs, West Grove, PA) in 5% FBS/PBS for an hour. Plates were washed with PBS and 3,3′,5,5′-tetramethylbenzidine (TMB substrate, Thermo Fisher Scientific, Waltham, MA) was added to each well. The reactions were stopped by adding 0.5 M HCl and the optical density was measured at 450 nm with a plate reader.

The 96-well ELISA plates (Thermo Fisher) were coated with 100 ng recombinant αS proteins in 100 μl PBS per well. Wells were washed with PBS and blocked with TBS/5% skim milk powder. Primary antibodies were added to blocking solution and incubated at room temperature. After TBS washes, plates were incubated with horseradish peroxidase-conjugated anti-mouse antibody (Jackson Immuno Research Labs) in TBS/5% skim milk powder for 30 min. Plates were washed with TBS and 3,3′,5,5′-tetramethylbenzidine substrate (Thermo Fisher Scientific) was added to each well. The reactions were stopped by adding 0.5 M HCl and the optical density was measured at 450 nm with a plate reader.

96-well ELISA plates (Corning Life Sciences, Corning, NY) were coated with 100 ng peptide in 100 µL phosphate buffered saline (PBS) per well using the peptide used for immunization (see Table 1). Wells were washed with PBS and blocked with PBS/5% fetal bovine serum (FBS). Primary antibodies were added to blocking solution and incubated at room temperature. After PBS washes, plates were incubated with horseradish peroxidase-conjugated anti-mouse antibody (Jackson Immuno Research Labs, West Grove, PA) in 5% FBS/PBS for an hour. Plates were washed with PBS and 3,3',5,5'-tetramethylbenzidine (TMB substrate, Thermo Fisher Scientific, Waltham, MA) was added to each well. The reactions were stopped by adding 0.5 M HCl and the optical density was measured at 450 nm with a plate reader.