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Combination of fluorescent ligand binding and immunohistochemistry makes it possible to simultaneously track down ligand and receptor following neuropeptide binding and internalization. Intracellular trafficking of ligand can also be monitored through combined visualization of the fluorescent ligand and of specific markers of intracellular compartments. Because significant amounts of ligand are lost in the course of immunohistochemical processing, this type of study is best performed in transfected cells as these express high concentrations of receptors and thus bind and internalize commensurately large amounts of fluorescent ligand. Presumably, the same type of approach should be applicable to cells or tissue slices expressing endogenous receptors, provided that the ligand is cross-linked to the receptor prior to immunohistochemical processing.
encoding either native or epitopetagged receptors.
ligand.
itself or against the immunogenic epitope in the case of epitope-tagged receptors; or antibodies directed against compartment-specific cellular antigens (e.g., rab proteins, lamp proteins, etc.).
tagged secondary antibodies.
as the secondary antiserum.
NaCl in 0.1 M phosphate buffer, pH 7.4.
1. Plate the transfected cells on 12-mm polylysine-coated glass for 1 to 2 hours at 37°C.
2. Incubate the transfected cells with the fluorescent ligand (20 nM) for various periods of time at 37°C as described above and rinse 3 times in ice-cold Earle’s buffer.
3. Fix the cells with 4% PFA for 20 minutes at room temperature.
4. Rinse twice with PBS.
5. Preincubate the cells for 20 minutes in PBS containing 3% normal serum.
6. Incubate for 60 minutes at room temperature with appropriate dilution of primary antibody in PBS containing 1% normal serum and 0.02% Triton X-100.
7. Rinse 3 × 5 minutes with PBS.
8. Incubate with the FITC-tagged secondary antibody diluted 1:100 to 1:500 in PBS for 30 minutes at room temperature.
9. Rinse 3 × 5 minutes in PBS.
10. Mount the coverslips, cell-side down on glass slides with Aquamount and examine by confocal microscopy. FITC signal is imaged by exciting samples with 488 nm and Bodipy red signal by exciting samples with 568 nm.
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