Peroxidase conjugated donkey anti rabbit igg

Whole cell lysates from DA and DA^Mut rats were obtained from brain samples by lysis in RIPA buffer (ThermoScientific). Samples were run in a NuPAGE 4–12% Bis-Tris gel (Novex, Invitrogen) and transferred to a PVDF membrane (EMD, Millipore). Blots were blocked with 5% bovine serum albumin (Sigma-Aldrich) in Tris-buffered saline with 0.1% Tween 20 (Cell Signaling Technology) and subsequently incubated with mouse anti-rat Endophilin A2 (clone S51-1, Origene Technologies, Inc) and rabbit anti-histone 2B (Cat. No C49810, LifeSpan Biosciences, Inc). Blot was incubated with peroxidase-conjugated goat anti-mouse IgG(Cat no. 115-035-062, Jackson ImmunoResearch laboratories Inc.) or peroxidase-conjugated donkey anti-rabbit IgG (Cat no.771-036-152, Jackson ImmunoResearch laboratories Inc.) respectively and developed using enhanced chemiluminescence. Pictures including the entire blot can be found in the Source Data file.

Blood serum was collected prior to surgery and NP vaccine injection, and at 0, 2, 4, 8 and 12 weeks after these procedures to detect the presence of anti-NP antibody responses using an NP ELISA custom created for this study. Briefly, a 96-well Immulon plate (ThermoFisher) was coated with 100 μL of NP protein isolated from pooled sheep NP material collected at necropsy. The NP protein was diluted to a concentration of 20ug/mL in carbonate buffer. After incubation overnight at 4°C, wells were washed with PBS Tween using a plate washer, and non-specific binding sites were blocked for 2 h at room temperature with PBS + 10% BSA. After washing with PBS, 100 μL of rabbit serum (diluted 1:100 in 1%BSA/PBS) were added to the plates and incubated for 2 h at room temperature. After a second plate wash, 100 μL of 1:3000 dilution (in PBS + 1% BSA) of peroxidase conjugated donkey anti rabbit IgG (Jackson Immuno Research) was added and incubated for 1 h at room temperature. Following washing with PBS, TMB-ELISA Substrate Solution (ThermoFisher) was added and incubated for 10 min. Finally, 50 μL TMB stop solution was added and the optical density (OD) was measured at an absorbance at 450 nm. Optical density values were plotted, and pre-vaccination serum ODs were compared to post-vaccination ODs.