The fate of internalized ligands is monitored by confocal microscopy following the labeling of cells in culture or of brain slices ex vivo with nanomolar concentrations of fluorescent derivatives of either native or metabolically stable analogs of peptide ligands. The distribution of the label may be analyzed either immediately after ligand exposure, as described below for studies in cell cultures, or after varying periods of chasing with physiological buffer, as described below for studies in brain slices.
encoding the appropriate receptors (for details on transfection procedure see Reference 17) or:
neonatal rat brain prepared as previously described (18,22).
α-Bodipy-[D-Trp8]somatostatin (fluo- SRIF), α-Bodipy-dermorphin, α-Bodipy- deltorphin. These fluorescent compounds were originally synthesized and purified for us by Dr. J.-P. Vincent (University of Nice-Sophia Antipolis, France). They are currently available from NEN Life Science Products.
(25 μg/mL polylysine, 15 min at room temperature) (Sigma, St. Louis, MO, USA).
1-piperazineethanesulfonic acid (HEPES) buffer, pH 7.4, containing 140 mM NaCl, 5 mM KCl, 1.8 mM CaCl2, 3.6 mM MgCl2 (all salts are from Sigma).
buffer containing 0.1% bovine serum albumin (BSA), 0.01% glucose, and 0.8 mM 1,10-phenanthroline (peptidase inhibitor), pH 7.4.
acid and 0.5 M NaCl in Earle’s buffer, pH 4.0.
PA, USA).
1. For experiments on transfected epithelial cells, plate the cells as a monolayer on 12-mm polylysine-coated glass coverslips and let them adhere for 1 to 2 hours at 37°C. For experiments in primary cultures, plate the cells onto polylysine-coated glass coverslips and allow them to grow in a humidified atmosphere at 37°C and 5% CO2 until fully differentiated (6–10 days).
2. Preincubate the cells for 10 minutes at 37°C in supplemented Earle’s buffer.
3. Incubate the cells for various periods of time (5, 10, 15, 30, 45, and 60 min) with 10 to 20 nM of the appropriate fluorescent ligand in supplemented Earle’s buffer. For determination of nonspecific labeling, add a hundredfold concentration of nonfluorescent peptide or antagonist to the incubation medium.
4. At the end of the incubation, rinse the cells 3 times with ice-cold Earle’s buffer or with hypertonic acid buffer to dissociate surface-bound ligand. At this point, cells may be fixed with 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. The latter procedure offers the advantage of allowing for coimmunolocalization of cellular antigens (see below).
5. Air-dry the cells rapidly and mount them up on glass slides with Aquamount. It is imperative that the cells themselves not be exposed to an aqueous medium (unless they were fixed) as this would promote dissociation of receptor– ligand complexes.
6. Examine by confocal microscopy. Images may be acquired as single midcellular optical sections or through multiple serial Z levels at 32 scans per frame.
The protocol described below was used to monitor the fate of internalized NT in slices from rat ventral midbrain tegmentum (11) and basal forebrain (10). Materials and Reagents
NaHCO3, 1.25 mM KH2PO4, 1.3 mM MgSO4, 5 mM KCl, 10 mM glucose, and 2.4 mM CaCl2.
Microscopy Science, Fort Washington, PA, USA): 4% PFA in 0.1 M phosphate buffer, pH 7.4.
in 0.1 M phosphate buffer, pH 7.4.
in 0.1 M phosphate buffer, pH 7.4.
1. Following decapitation of the rat, rapidly remove and immerse the brain in a cold oxygenated (95% O2, 5% CO2) Ringer buffer.
2. Cut 300 to 400-μm-thick slices through the regions of interest with a Vibratome.
3. Equilibrate the slices for 45 minutes in oxygenated Ringer buffer at room temperature.
4. Superfuse the slices for 3 minutes at 37°C with 20 to 40 nM fluorescent ligand in Ringer buffer.
5. Rinse with oxygenated Ringer buffer for 5, 10, 15, 30, 45, or 60 minutes at 37°C. To control for nonspecific labeling, incubate additional slices in the presence of 100- to 1000-fold excess of nonfluorescent probe or antagonist.
6. After rinsing, fix the slices for 30 minutes at room temperature with 4% PFA.
7. Immerse the slices overnight in the cryoprotectant solution, flatten on tissue chuck, snap freeze in isopentane at -60°C, and resection at 45 μm thickness in the plane of the slice on a freezing microtome.
8. Mount frozen sections on gelatin-coated glass slides with Aquamount and examine by confocal microscopy.
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