Horseradish peroxidase conjugated goat anti rabbit antibody

Recombinant MVA-gBΔfur731 and wtMVA virus titers were performed on DF-1 cells. DF-1 cells were seeded at 4x10⁵ cells/well in 24-well plates, infected with 100μL of 10-fold serial dilutions of virus stock for 2 hours, overlaid with growth media containing 0.5% methylcellulose, and incubated for 2 days at 39°C. After incubation, cells were washed with PBS, fixed with 1:1 methanol:acetone, and blocked with PBS/4% FBS for 1 hour. Rabbit anti-vaccinia antibody (Invitrogen, PA1-7258) was diluted in PBS/4% FBS (1:500) and incubated with cells for 1.5 hours at room temperature (RT). After washing with PBS and PBS/4% FBS, goat anti-rabbit horseradish peroxidase conjugated antibody (Jackson ImmunoResearch Laboratories Inc., 111-035-144) was diluted in PBS/4% FBS (1:500) and incubated with cells for 45 minutes at RT. ImmPACT DAB Peroxidase Substrate (Vector Labs, SK-4105) was diluted in appropriate kit buffer and developer was added to cells for 10 minutes. Plates were washed twice with H₂O, dried overnight, and plaque spots counted for quantification.

Western blot analysis of C2C12 myoblasts was performed as described (Micheli et al., 2011 (link)). Briefly, cells were lysed by sonication in buffer containing 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.2% Nonidet P-40, with protease inhibitors 1 mM Na₃VO₄, 10 mM 2-glycerophosphate, 10 mM NaF, 5 mM ATP, 5 mM MgCl₂. Proteins were electrophoretically separated by SDS-PAGE and transferred to nitrocellulose filters. Immunoblots were performed hybridizing filters to a rabbit polyclonal antibody against Id3 (Santa Cruz Biotechnology; Sc-490, 1:300); detection of the second antibody (goat anti-rabbit horseradish peroxidase-conjugated antibody; Jackson ImmunoResearch) was performed by chemiluminescent assay.