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INTRACELLULAR NEUROBIOTIN DYE FILLS-NUSBAUM LAB PROTOCOL (Feb. 16, 1996) SUPPLIES: 1. Neurobiotin- Vector Labs; Cat. #SP-1120; size- 50 mg; price- $45. Vector Laboratories, Inc. 30 Ingold Road Burlingame, CA 94010 USA (Phone: 415-697-3600) (FAX: 415-697-0339) 2. FITC-avidin D- Vector Labs; Cat. #A-2001; size- 5 mg; price- $60. 3. FITC-anti-avidin antibody- Vector Labs; FITC-Anti-Avidin D antibody, Affinity Purified. Cat. #SP-2040; size- 0.5 mg active component; produced in: Goat; price- $50. [We used to use a Sigma antibody, but switched to Vector Labs antibody just to see if it was better. I am not certain that it is better, but it certainly is not worse. Here is the original Sigma info: Sigma; Cat. #F-1269: Mouse Monoclonal Anti-Avidin, FITC conjugate; size- 0.5 ml; price- $80. SOLUTIONS: 1. Neurobiotin: 10% in H2O 2. FITC-avidin D: Working dilution- 1:5,000 in P-triton. 3. FITC-anti-avidin antibody: Working dilution- 1:5,000 in P-triton. 4. FIX: 4% Paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.3) 5. P-triton: 0.3% Triton X-100 in 0.1 M sodium phosphate buffer (pH 7.3) 6. P: 0.1 M sodium phosphate buffer (pH 7.3) 7. Mounting Medium: 80% glycerol and 20% 20 mM sodium
carbonate (pH 9-9.5) INTRACELLULAR NEUROBIOTIN DYE FILLS-NUSBAUM LAB PROTOCOL (Feb 16, 1996) PROCEDURE: 1. Pull a normal intracellular recording-type microelectrode. Fill the tip of the electrode with the Neurobiotin solution. Then leave an air space and fill the rest of the electrode with 3.0 M KCl. 2. Iontophoresis: Depolarizing current is necessary. We use the same type of procedure as for LY, except for the use of positive (+) current. So: Frequency- 1 Hz; Duration- 500 msec; Amplitude- +5nA; Filling Time- 1 to 2 hours (2 hours is preferable). OR Pressure Injection (w/0.25% Fast Green): Use 2-5 PSI, 1 sec duration pulses until the soma becomes a deep green. This usually takes us 5-10 injections, separated by several minutes each to allow soma swelling to subside. These injections are way faster than the iontophoresis (by nearly 2 hours) but they do not label neuropil as well. Recently, we have quit using pressure and returned to iontophoresis. 3. At the end of the experiment, fix the prep overnight in 4% paraformaldehyde, at 4oC. [NOTE: Alternatively, for long distance fills, leave the prep in the fridge overnight before fixation, to optimize dye-spread.] 4. All incubations should be done at 4oC. 5. Move the preparation to a small vial. Rinse the prep with P-triton 5 times, at intervals of at least 1 hour. Each rinse can last for more than 1 hour, but not less. Rinses can also continue overnight, but do 5 rinses total. 6. After the final rinse, incubate the prep in FITC-avidin D for 18-24 hours. 7. Next, rinse the prep another 5 times with P-triton. 8. Next, incubate the prep in FITC-anti-avidin antibody for 18-24 hours. 9. Next, rinse the prep another 5 times, but use P instead of P-triton. 10. Then, mount the prep, take it to the fluorescence microscope and be happy. NEUROBIOTIN BACKFILLS- Nusbaum Lab Procedure (5-24-96) SUPPLIES: 1. Neurobiotin- Vector Labs; Cat. #SP-1120; size- 50 mg; price- $45. Vector Laboratories, Inc. 30 Ingold Road Burlingame, CA 94010 USA (Phone: 415-697-3600) (FAX: 415-697-0339) 2. FITC-avidin D- Vector Labs; Cat. #A-2001; size- 5 mg; price- $60.
SOLUTIONS: 1. Neurobiotin: 5% or 10% in H2O 2. FITC-avidin D: Working dilution- 1:5,000 in B-triton. 3. FITC-anti-avidin D antibody: Working dilution- 1:5,000 in B-triton. 4. FIX: 4% Paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.3) 5. B-triton: 0.3% Triton X-100 in 50 mM sodium bicarbonate buffer, with 150 mM NaCl (pH 8.0-8.5) 6. B: 50 mM bicarbonate buffer, with 150 mM NaCl (pH 8.0-8.5) 7. Mounting Medium: 80% glycerol and 20% 20 mM sodium carbonate (pH 9-9.5) NEUROBIOTIN BACKFILLS- Nusbaum Lab Procedure (5-16-95) 1. (A) Build a vaseline well around the relevant nerve. (B) Remove saline from the well and determine if the well leaks. (C) RInse the well twice with deionized or distilled water, then remove the water and add the Neurobiotin solution (10% in distilled water). (D) CUT THE NERVE. (Do not forget this step) (E) Label the lid of the prep dish, then incubate the prep @4oC for approximately 48 hours. 2. (A) After 48 hours, replace the Neurobiotin solution in the well with normal saline. (B) Remove the vaseline and pin down the loose endings of the cut nerve. (C) Carefully pour out the saline and pour in a solution of 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.3). (D) Incubate the prep in fix for approx. 12-24 hours, @4oC. (E) All subsequent steps should also occur @4oC. 3. (A) Rinse out the fix with several changes of sodium bicarbonate buffer (50mM, pH 8.0-8.5). (B) Cut short all of the peripheral nerves and unpin the prep. (C) Move the prep into a small vial containing bicarbonate buffer with 0.3% Triton X-100. (D) Rinse 5 times (approx. once every hour) with the buffer plus Triton X-100. 4. Incubate the prep for 18-24 hours with FITC-cojugated Avidin D, at a final dilution of 1:5,000. Dilution should be made with buffer plus Triton X-100. 5. Rinse 5 times (approx. once every hour) with the buffer plus Triton X-100. 6. Incubate the prep for 18-24 hours with FITC-conjugated anti-Avidin D antibody, at a final dilution of 1:5,000. Dilution should be made with buffer plus Triton X-100. 7. Rinse 5 times (approx. once every hour) with the buffer (no Triton X-100 used here). 8. Mount the prep with 80% glycerol plus 20% 20 mM sodium carbonate (pH 9.0-9,5).
Best of luck! Michael P. Nusbaum |
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