Systematic Veterinary Virology

PROCEDURE

• Grow cells on sterile glass cover slips or slides overnight in 37^oC/5% CO₂ incubator
• Rinse briefly with PBS
• Fix cells by incubation with 1% formalin in PBS for 10 minutes
• Rinse three times with PBS
• Incubate for 10 minutes with 0.1% H₂O₂ in PBS to quench endogenous peroxidase activity
• Rinse once with PBS
• Incubate slides for 20 minutes in 10% goat serum in PBS (suppresses non-specific binding of IgG)
• Wash with PBS
• Incubate with primary antibody for 1 hour at room temperature or overnight at 4^oC. Optimal antibody concentration is usually from 1-10ug/ml in PBS-BSA
• Wash three times with PBS
• Incubate with biotin-conjugated secondary antibody for 45 minutes. Again, optimal antibody concentration is usually from 1-10ug/ml in PBS-BSA
• Wash three times with PBS
• Incubate with streptavadin-HRP for 15 minutes. This step should be titrated as excess streptavadin-HRP can lead to high background
• Wash with 0.5% triton x-100 in PBS for 30 seconds
• Incubate with fresh DAB solution + (substrate - H2O2/Urea) for 5 minutes. This step can be lengthened if additional sensitivity is necessary. It is a good idea to do a quick spot check here
• Rinse with distilled water
• Counterstain with hematoxylin
• Dehydrate with alcohol and xylene
• Mount permanent coverslip