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General Dye Filling Protocol (for more detail, see: http://stg.rutgers.edu/resources/IntraStain.pdf

  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 LY and Alexa-488: (can use aliquot for up to a week, keep in the fridge in the dark otherwise)

    1. thaw aliquot

    2. for Alexa-488: if making 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. quick spin on mini-centrifuge

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

    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 1-2ul 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)) (LY has a negative charge, so negative pulses should push dye into neuron)

    1. During the 0nA step, monitor Vm

    2. Also monitor current through the electrode; if 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) (LY has a negative charge, so negative pulses push the dye into the neuron)

  5. After injecting dye, let 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. The website address is: http://neuromorpho.org/WIN.jsp


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