Rabbit polyclonal anti neurofilament 200 antibody

The quantitative assessment of the intensity of neurodegeneration was performed using the ELISA method with primary antibodies directed against phosphorylated neurofilaments. The first step was the coating of a 96-well Maxisorb Microtitre plate (Nunc, Roskilde, Denmark) with monoclonal anti-NfH antibodies (SMI35R; Sternberger Monoclonals; Convance Princeton, NJ, USA) and followed by overnight incubation at 4 °C. Primary antibodies were diluted in carbonate buffer, pH 9.6. The next day, the samples and standard protein (neurofilament, 200 kD; Progen, Heidelberg, Germany) were added. As a secondary antibody, rabbit polyclonal anti-neurofilament 200 antibody (Sigma-Aldrich, Poznan, Poland) was used. As a tertiary antibody, swine antibodies against rabbit immunoglobulin conjugated with horseradish peroxidase (Dako, Glostrup, Denmark) were used. The final step was the addition of a color substrate for horseradish peroxidase, which was 3,3′,5,5′-tetramethylbenzidine (Sigma-Aldrich, Poznan, Poland). To stop the color reaction, 1 M HCl was used and the color photometric assessment was done using a VICTOR2 Wallac 1420 reader (PerkinElmer, Waltham, MA, USA). The analysis was performed at a 450 nm wavelength, corrected at a 595 nm wavelength. All samples were analyzed in duplicates and the concentration of NfH was determined by referring to the standard curve.