Procedure
1. Dissolve 200 µg of FITC in 200 µl of conjugation buffer and immediately mix it with 1 ml of streptavidin solution.
Note: When substituting TRITC for FITC, a similar protocol can be followed; however, the TRITC must first be
dissolved in dimethylsulfoxide (DMSO) at 100 µg/100 µl instead of 200 µg/200 µl in conjugation buffer.
2. Incubate for 1 hour at 37°C in the dark.
3. Remove excess and hydrolyzed FITC by gel filtration.
Procedure for Labeling an Antibody with FITC
Materials Required
• Conjugation buffer: 100 mM carbonate/bicarbonate buffer, pH 9.0
• Antibody solution: dissolve ~1 mg of antibody in 1 ml of conjugation buffer
• Zeba Desalt Spin Column (Product No. 89891) or other gel filtration column with a 5,000-7,000 MW cut-off
Procedure
1. Dissolve FITC in conjugation buffer at a final concentration of 1 mg/ml immediately before use.
2. Add 10 µl of FITC solution to the 1 ml of the antibody solution; mix thoroughly.
3. Incubate for 1 hour at room temperature in the dark.
4. Remove excess and hydrolyzed FITC by gel filtration.
Procedure for Labeling an Antibody with TRITC
This method is adapted from Larsson.
2
Materials Required
• TRITC: dissolve in DMSO at 1 mg/ml
• Conjugation buffer: 100 mM carbonate/bicarbonate buffer, pH 9.0
• Antibody solution: dialyze antibody into conjugation buffer at 6 mg/ml
• Zeba Desalt Spin Column (Product No. 89891) or other gel filtration column with a 5,000-7,000 MW cut-off
Procedure
1. While stirring, slowly add 35 µl of TRITC to 1 ml of the 6 mg/ml antibody solution; mix thoroughly.
2. Incubate for 2 hours at room temperature in the dark.
3. Remove excess and hydrolyzed TRITC by gel filtration.
Additional Information
• Visit the web site for a listing of related Thermo Scientific products (other fluorophores and convenient labeling kits) and
technical resources such as the Tech Tip: Calculate dye:protein (F/P) ratios.
• Fading (photobleaching in tissue sections) can sometimes be reduced by mounting in an alkaline buffered media (pH 9).
2
There are several reagents that may be used with FITC and/or TRITC derivatives to prevent fading including n-propyl
gallate at 0.1-0.25 M dissolved in glycerol for FITC or TRITC.
3
For FITC derivatives, o- or p-phenylenediamine added
to the mounting buffer from 1 µg/ml to 1 mg/ml in glycerin also may be used.
2,3
References
1. Horisberger, M. (1984). In Immunolabeling for Electron Microscopy. Polak, J., Varndel, I. Ed. Elsevier: Amsterdam, p. 98.
2. Larsson, L. (1988). Immunocytochemistry: Theory and Practice. CRC. Boca Raton, 77-83, 224-225.
3. Goding, J. (1986). Monoclonal Antibodies: Principles and Practice, 2nd ed. Academic, London.