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The present technique applies standard pre-embedding immunogold procedures, as originally developed in V. Pickel’s laboratory (5), to the electron microscopic detection of neuropeptide receptors in transfected epithelial cells, primary neuronal cultures, or brain slices, following stimulation by an unlabeled agonist. As for confocal microscopic studies, labeling may be carried out either by pulse chase as described below for studies in cell culture, or immediately after stimulation with the agonist, 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). To Be Prepared Fresh on Day 1
(SPB), pH 7.4: 154 mM Na2HPO4, 23 mM NaH2PO4H2O. Do not adjust pH.
7.4: 1.2% (wt/vol) Trizma base, 0.9% NaCl. Adjust to pH 7.4 with HCl.
Science) and 2% PFA in 0.1 M SPB.
(Sigma) from the same species as secondary antibody diluted in 0.1 M TBS.
X-100 and 0.5% normal serum in 0.1 M TBS.
the receptor itself or an epitope tag and diluted in antibody dilution buffer (dilution must be worked out for each antibody).
KCl, 1.8 mM CaCl2, 0.9 mM MgCl2*6 H20, 25 mM HEPES.
in concentrations ranging from 10 nM to 10 μM in binding buffer consisting of 0.8 mM 1,10-phenanthroline, 0.1% D-glucose, 1% BSA in Earles’ buffer. To be Prepared Fresh on Day 2
water and 0.9% NaCl. Adjust to pH 7.4.
gelatin stock and 8.0% (wt/vol) BSA in 0.01 M PBS.
Science) in 0.01 M PBS.
antibodies directed against species in which primary antibody was raised (IgG-gold conjugate; Amersham Pharmacia Biotech, Little Chalfont, Bucks, England, UK), diluted 1:20 in washing incubation buffer.
sodium citrate (trisodium citrate, dehydrated) in double-distilled water; adjust to pH 7.4 with 0.2 M citric acid (2.1 g in 50 mL distilled water).
M SPB (prepare immediately before use and keep in the dark at all times).
Pharmacia Biotech).
These experiments are carried out on cells that have been cultured directly into the bottom of plastic culture dishes (21).
Incubate cells with desired concentration of agonist. For pulse-chase labeling over multiple time points: (i) preincubate in binding buffer for 5 minutes at 4°C; (ii) pulse with agonist dissolved in binding buffer for 30 minutes at 4°C; and (iii) chase with binding buffer for various time points at 37°C.
1. Fix with 2% acrolein in 2% PFA for 20 minutes at room temperature.
2. Post-fix with 2% PFA for 20 minutes at room temperature.
3. Rinse 2 × 10 minutes in 0.1 M TBS.
4. Incubate in blocking buffer for 30 minutes.
5. Incubate overnight at 4°C with appropriate dilution of primary antibody directed against the receptor in antibody dilution buffer.
1. Rinse 3 × 10 minutes in 0.01 M PBS.
2. Rinse for 10 minutes in washing incubation buffer.
3. Incubate for 2 hours at room temperature with the IgG-gold conjugate.
4. Rinse for 5 minutes in washing incubation buffer.
5. Rinse 3 × 5 minutes in 0.01 M PBS.
6. Fix 10 minutes with 2% glutaraldehyde.
7. Rinse for 5 minutes in 0.01 M PBS.
8. Rinse twice in 0.2 M citrate buffer.
9. Silver intensification: mix solutions A and B in each well and develop for 7 minutes.
10. Rinse twice in 0.2 M citrate buffer.
11. Rinse for 10 minutes in 0.1 M SPB.
12. Postfix for 10 minutes in 2% osmium tetroxide (in the dark).
13. Dehydrate in graded ethanols: 50% EtOH for 2 × 5 minutes. 70% EtOH for 2 × 5 minutes. 80% EtOH for 5 minutes. 90% EtOH for 5 minutes. 95% EtOH for 10 minutes. 100% EtOH for 2 × 15 minutes.
14. Embed in Epon as follows: a. Apply 1:1 Epon: propylene oxide solution for 1 minute and aspirate. b. Apply 1:3 Epon: propylene oxide solution for 3 minutes and aspirate. c. Apply one drop of 100% Epon to the surface of the cells.
15. Place the flush surface of the cylindrical plastic mold onto the bottom of the well. Ensure a good seal between the mold and the bottom of the well, and that the mold is perpendicular to the bottom.
16. Carefully fill in the area between the inside surface of the culture plate and the plastic mold with plasticine modeling clay.
17. Fill the plastic mold with 100% epon.
18. Replace the 4-well plate cover, add lead weights to the lid, and cure in a 60°C oven for 13 to 16 hours.
19. Remove the plasticine modeling clay and crack off the polymerized Epon blocks from the surface of the culture plate.
20. Examine the bottom surface of the polymerized Epon block under the dissecting microscope for labeled cells.
21. Trim the block around the labeled cells.
22. Incubate in a 60°C oven for at least another 24 to 48 hours before cutting with the ultramicrotome.
(200–250 g).
KCl, 1.2 mM NaH2PO4, 2.4 mM CaCl2, 1.5 mM MgSO4, 26 mM NaHCO3, 10 mM glucose, pH 7.4.
in 0.1 M SPB, pH 7.4.
3% glycerol.
reagents and buffers as listed for studies in cell cultures.
1. Decapitate the rats and rapidly remove the brains.
2. Block and section the region(s) of interest on a vibratome and collect slices (100 μm) in ice-cold Ringer buffer, continuously oxygenated by a mixture of 95% O2 and 5% CO2.
3. Equilibrate slices in Ringer buffer for 40 minutes at room temperature.
4. Preincubate slices in Ringer buffer for 15 minutes at 37°C.
5. Incubate slices in Ringer buffer containing various concentrations of agonist for 10 to 60 minutes at 37°C.
6. Fix slices with fixative.
7. Rinse twice in 0.1 M SPB.
8. To permeabilize the tissue, incubate sections in cryoprotectant solution for 30 minutes, freeze in isopentane at -70°C, dip in liquid nitrogen, thaw in 0.1 M SPB at room temperature.
9. Immerse in blocking buffer for 30 minutes.
10. Incubate overnight at 4°C with appropriate dilution of receptor antibody in antibody dilution buffer.
1. Carry out immunolabeling with secondary antibody, postfixation in 2% OsO4 for 40 minutes, and dehydration of slices as described for cell cultures above (day 2, steps 1 through 13).
2. Then, embed slices as follows: a. Immerse in 1:1 Epon: propylene oxide solution for 30 minutes and aspirate. b. Immerse in 1:3 Epon: propylene oxide solution for 30 minutes and aspirate. c. Immerse in 100% Epon overnight at 4°C.
3. Flat-embed the sections in between two sheets of acetate film; lay on a flat, smooth surface, and add lead weights to the top.
4. Cure in a 60°C oven for 24 to 48 hours.
5. Gently remove one of the acetate sheets from the surface of the embedded tissue.
6. Add a thin film of cyanoacrilate glue to the surface of the embedded tissue and quickly affix to the flush bottom surface of a polymerized Epon block.
7. Trim the block around the labeled cells.
8. Cure at 60°C for at least another 24 to 48 hours before cutting with the diamond knife.
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