Dye Fills

General Dye Filling Protocol

For more detail, see the Intracellular Staining Protocol (below) from the NJIT STG Lab website.

1. Acquire dye to fill with (Lucifer Yellow and Alexa Fluor 488 are in the -20C Freezer, in Marie Injection Dyes//Adriane Injection Dyes and Injection Dyes - Adriane respectively).

2. For Lucifer Yellow and Alexa-488: (can use aliquot for up to a week, keep in the fridge in the dark otherwise).

   1. 1. Thaw the aliquot.

      2. For Alexa-488: if making a new dye solution, add 3-4 drops Scott-Hooper to 1 aliquot. Then continue with c and d.

      3. Vortex for a few seconds.

      4. A quick spin on the picofuge.

3. Attempt to backfill a low-resistance electrode (5-15 MOhms) with the dye only.

   1. 1. Test the resistance of the electrode in the bath–ideally, Relectrode should stay low even with the backfill ((no more than 40 MOhms or so; but ideally between 10-20 MOhms).

      2. If you are having trouble getting low resistance measurements, you can try unclogging the electrode by vortexing the dye a bit more and/or adding a maximum of 1-2μl electrode juice (vortexing, quick spin) to dissolve the dye.

      3. Try to stick with backfill alone; do not add internal to electrode after back-filling (this dilutes the dye).

4. To fill the cell, set up an episodic stimulation protocol with -4nA pulses (or -6nA pulses if you aren’t planning on doing anything besides immuno with the prep) at 1 Hz, 250ms 0nA step (off), 500 ms step (on), 250 ms 0nA step (off)) (Lucifer Yellow has a negative charge, so negative pulses should push dye into the neuron).

   1. 1. During the 0nA step, monitor Vm.

      2. Also, monitor the current through the electrode; if the pulse changes from a square wave to something noisier, it is probably clogged (so re-stick with a new electrode and try again).

      3. To fill the cell, set up an episodic stimulation protocol with -4nA pulses (or -6nA if you aren’t planning on doing anything besides immuno with the prep) at 1 Hz, 250ms 0nA step (off), 500 ms step (on), 250 ms 0nA step (off) (Lucifer Yellow has a negative charge, so negative pulses push the dye into the neuron).

5. After injecting the dye, let the cell sit for 1/2hr to let the dye diffuse (before fixing, etc.).

6. If you have a fluorescent lamp, you can visualize the cell with it, but be careful not to use it too much–the fluorescent lamp will cause the cell to undergo damage over time.

7. You can also use the Leica camera to visualize the fill–it can sometimes pick up smaller projections than you can with your own eyes and the oculars.

Neuro Morphology Site

This is a website with publically available reconstructions of different kinds of neurons. Follow the link to access the website: Neuro Morpho.