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Neurobiotin protocol and chemicals Source: Otto Friesen’s lab (with small revisions from Bill Kristan’s lab (6/13/95) CHEMICALS & SUPPLIERS Neurobiotin, Sold as Neurobiotin tracer Anti-biotin Jackson Immunoresearch Labs, Inc. Paraformaldehide Goat Serum Triton X-100 Glycerol Sodium bicarbonate Vectashield mounting medium (optional) Vector Labs. Inc OTHER SUPPLIES Glass slides Long cover slips Parafilm Small vials for frozen antibody aliquots and for neurobiotin solutions 10 ml tubes for storing frozen goat serum Large Petri dishes and covers for storing slides with mounted preparations Fingernail polish STOCK SOLUTIONS Solution A 0.1 M Na2HPO4. Make 4 vols Solution B 0.1 M Na2H2PO4. Make 1 vol.
PB 0.1 M phosphate buffer (combine solutions A and B in a ratio of 4:1; adjust pH to 7.3 to 7.4 by adding additional A or B). Make 1 lt PBX 0.1 M phosphate buffer with 0.3% Triton X-100 (make up five 50 ml bottles) Goat Serum Comes as frozen liquid; make aliquots of about 5 ml before freezing PBXG 0.1 M phosphate buffer with 0.3% Triton X-100 and 10% goat serum (make up five 50 ml bottles) KAc 2 M solution for intracellular electrodes (100 ml) Neurobiotin 4% Neurobiotin in 2 M KAc (4.7 mg neurobiotin in 100 :l of 2 M Kac) Glycerol For mounting. 80% glycerol with 20 mM sodium bicarbonate in dH2O FRESH SOLUTIONS (made up fresh for each use) Antibody Cy3-conjugated IgG fraction monoclonal mouse anti-biotin dilute 1:2000 in PBXG. Comes as a freeze dried powder. Upon arrival dissolve in 0.4 ml dH2O. Let stand for 1 hr or use gentle vortexing. Solution should be clear. Store frozen in 10:l aliquots. Paraformaldehyde: 4% solution (see separate protocol for paraformaldehyde preparation). Make two 100 ml bottles PROTOCOL 1. Backfill microelectrodes with 4% neurobiotin in 2 M Kac. Place electrodes upside down (point upward) into vial containing the neurobiotin; wait ~10 min, then verify that the tip has filled by looking for a meniscus near the electrode shank under a dissecting scope. 2. Inject neurobiotin into cells with 0. S duration, +3-4nA current pulses, repeating every 1 sec for about ½ hour. Clogging of the electrodes is reduced by applying a continuous negative current of about -0.1 nA while applying the current pulses. 3. Leave the preparation undisturbed for 1 hr to allow neurobiotin to diffuse to the processes of the injected neuron. 4. Fix the ganglion with the 4% paraformaldehyde solution for at leats 4 hrs (overnight is better). Keep dish covered, paraformaldehyde is nasty!! 5. Rinse 6 times (1 hr between rinses at 4oC or ½ hr at room temp) with PBX solution, changing the solution twice during each rinse. This is done with the prep still pinned in the dish. For the final rinse, use the PBXG solution. 6. Remove the PBXG solution and apply the antibody solution. Place on a tilt table to provide continuous, gentle agitation for at least 4 hrs (or overnight) at 4oC. 7. Rinse preparation 6 times during the course of 1 hr with PB solution. Place on tilt table between rinses. 8. To mount the preparation make a spacer of a single layer of parafilm around the edge of the glass slide. One easy way of doing this is to cut a square of parafilm the size of a cover slip. Then cut the center of the parafilm to leave a narrow border, which can be place on the glass slide. The parafilm serves as a spacer to prevent the cover slip from squashing the preparation. Transfer the preparation from the dish to the slide. Add the glycerol mounting medium. Place the cover slip over the preparation and paint the edges with nail polish to seal the edges. 9. View the injected cells with fluorescence microscope using the filters used to view rhodamine. |
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