Donkey anti goat igg hrp linked antibody

Confluent monolayers of MDCK cells in 12 well plates were infected with 20–50 plaque forming units (PFU) of virus for 1 h at 37 °C. The cells were washed thoroughly and overlaid with 1.2% of Avicel RC-591 (FMC Biopolymer) alone or with MAb 65, MAb 37, MAb 148 or isotype control (IgG1 + IgG2a) at 100 μg/mL supplemented with 2 μg/mL of TPCK-treated trypsin (Sigma). The cells were then incubated for 72 h at 37 °C in 5% CO₂. Avicel was subsequently removed and the cells were fixed with 4% PFA for 15 min. After permeabilization (10x Permeabilization buffer diluted in bi-distilled water, eBioscience), the cells were stained with 1/2000 diluted polyclonal goat anti-influenza ribonucleoprotein (RNP) (Biodefense and Emerging Infections Resources Repository, NIAID, NIH, NR-4282) followed by donkey-anti-goat IgG HRP-linked antibody (Santa Cruz Biotechnology, cat no. SC2020). After washing, TrueBlue peroxidase substrate (KPL) was used to visualize the plaques. The wells were also scanned and the plaque size was determined using ImageJ Analysis Software.

Paraffin embedded sections were detected with Sost antibody (anti-goat; R&D systems, AF1589). Antigen retrieval was performed by treating the sections with Proteinase K (20 μg/ml, Roche) at 37 °C for 15–20 minutes. Sections were then treated with 3% hydrogen peroxide for 10 minutes followed by blocking with 5% BSA and 10% donkey serum in 1X PBST. Sections were incubated with the Sost antibody at 1:100 dilution overnight, then donkey anti-goat IgG-HRP linked antibody (Santa Cruz Biotechnology) at 1:1200 for 1 hour and developed using DAB chromogen (Life Technologies). Three to five images were taken along the mid-diaphyses of femur, starting from 2 mm proximal to the growth plate, and Sost positive cells were quantified.