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Lucifer Yellow
Solutions: (For how to make solutions look here) 1) 5-10% LY solution in bidestilled water (10% is better) Cell Fills: Prepare a 5-10% LY solution in bidestilled water (Sigma’s is good; Molecular Probes’ sometimes works less well). Use microelectrodes that will give ~15MOhm in 3M KCl. Fill th tip with the LY solution and backfill the rest of the pipette with 1M LiCl. Inject iontophoretically at 1Hz (50% duty cycle) with -3 to -5nA for ~20-30min and then let the cell sit for another 20-30min for LY to diffuse. Then fix, rinse, mount, etc. (see below) Backfills: Prepare a 10 to 15% LY solution in bidestilled water. Make a vaseline well around the desired nerve, rinse it with water (to get rid of potassium which will precipitate the LY) , put the LY in the well and cut the nerve across. Wait 12 to 24hrs at 4 to 10o C, rinse the well several times with water and fix the prep in paraformaldehide overnight at 4o C or for 5-6hrs at room temp, rinse with phosphate buffer 5-6 times for 1 hr each at 4o C, or ½ hr at room temp. Then either proceed with immunohistochemistry or mount the prep to view under fluorescence. Fix: At the end of filling or backfilling fix the prep in the dish (to preserve shape) in 4% Paraformaldehyde (made in 0.1 M sodium phosphate buffer, pH 7.3) overnight at 4oC. [NOTE: Alternatively, for long distance fills, leave the prep in the fridge overnight before fixation, to optimize dye-spread.] All incubations should be done at 4oC !!! After fixation, move the preparation to a small vial. Rinse the prep with P buffer 6 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 6 rinses total. Keep notes of everything you do! Then, mount the prep. Mounting Medium: 80% glycerol and 20% 20 mM sodium carbonate (pH 9-9.5). Add a drop of this solution, which is very viscous, onto a microscope slide, put the prep in the drop, stretch it out and finally cover it with the coverslip. Take it to the fluorescence microscope and be happy. |
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