Multi-color immunofluorescence microscopy in cultured cells

Abstract

Immunofluorescence microscopy is a powerful method for analysis of the subcellular localization of the protein of interest. The use of fluorescence is very effective for multiple labeling and for higher magnification observation with a laser confocal microscope. A basic protocol of the immunofluorescent staining in cultured cells with some useful suggestions are introduced in the present paper. The author describes the following contents. 1) Coverslips: coverslips are necessary to be coated with coating reagent such as collagen to improve the attachment and growing of the cells. Commercially available glass slides for cell culture are also useful. Permeable support filters are convenient for establishing the apical and basolateral compartments when epithelial cells are cultured. 2) Fixation: basic fixative is 4% paraformaldehyde in phosphate buffer. Ethanol including 1% acetic acid or pure ethanol or methanol are also effective for some antigens. 3) Permeabilization: treatment with Triton X-100 or saponin before and sometimes during the antibody incubation is required to improve the antibody accessibility. 4) Blocking: nonspecific binding of antibodies is blocked with 5% serum from the animal species as same as that of the secondary antibody you use. 5) Antibody incubations: antibodies are diluted in the blocking solution and incubated with samples. For multi-labeling with primary antibodies derived from different animal species, the specimen can be sequentially incubated with a mixture of primary antibodies and a mixture of secondary antibodies. 6) Choice of secondary antibodies: secondary antibodies which do react specifically to the target without cross-reaction and appropriate fluorescent dyes for multiple labeling are required. 7) Sample storage: the fluorescence can be kept for long period like several months to years in specimens which are mounted with anti-fading mounting medium and stored at −20°C. 8) Trouble shootings: some trouble shootings are also shown. The author hopes that this paper would help the readers to obtain better results in immunofluorescence.