Protocol for Immunofluorescence staining of tissue culture cells

1. Grow cells on sterile glass cover slips or slides overnight in 37^oC/5% CO₂ incubator
2. Rinse briefly with PBS
3. Fix cells by incubation with 1% formalin in PBS for 10 minutes
4. Rinse three times with PBS
5. Incubate slides for 20 minutes in 10% goat serum in PBS (suppresses non-specific binding of IgG)
6. Wash with PBS
7. 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
8. Wash three times with PBS
9. Incubate with biotin-conjugated secondary antibody for 45 minutes. Again, optimal antibody concentration is usually from 1-10ug/ml in PBS-BSA
10. Wash three times with PBS
11. Incubate with streptavadin-fluorescein or streptavadin-texas red for 15 minutes in a dark chamber. This step should be titrated as excess streptavadin-label can lead to high background
12. Wash a minimum of three times with PBS
13. Mount aqueous coverslip

Notes: Incubations are to be at room temperature or a 4^oC humidified chamber. Storage is in the dark at 4^oC. Antibody concentrations here may not hold for all sources. These concentrations should be titrated for each source.