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When performing microelectrode recordings, it is critical to have some means of reconstructing the path of the electrode. One way to do this is to combine traditional electrolytic lesioning with fluorescent labeling of the electrode with a dye such as Di-I.
Specific hazards:
Di-I is an extremely effective fluorescent label for biological tissue. Gloves must be worn when measuring and Di-I powder and coating the electrode.
Chloroform is a liquid that evaporates rapidly and is an irritant. Chloroform must be stored under the fume hood with the lid screwed on. When withdrawing chloroform, work in the fume hood with the sash low. Open the lid only briefly and use a glass pipette with rubber bulb to withdraw a few drops of chloroform, and then recap the lid. Work with paper towels that can be disposed of easily after any waste chloroform evaporates.
Equipment hazard: the microelectrode amplifiers can be damaged by the currents employed with electrolytic lesioning. Make sure the preamplifiers or amplifiers are disconnected before lesioning.
Di-I labeling Procedure:
Prepare a paper towel in the fume hood. Place a few milligrams of Di-I crystals (maybe 2-5mg, no need to measure, just use a small spatula to scoop a little out of the Di-I container) in the center of a glass slide. Place an electrode on the slide so the the tip is near the pile of Di-I crystals. Be careful not to touch the tip of the electrode against anything so the fragile tip will not be damaged; note that the electrode can lay gently on the glass slide without being damaged. Using a glass pipette with a rubber bulb, place a small drop of chloroform on the Di-I crystals. The crystals will rapidly dissolve and flow onto the slide and the electrode. Wait about 30 seconds until the chloroform evaporates. The electrode tip area should appear bright red and be coated with Di-I.
Some materials needed: on a paper towel, there is an electrode placed gently on a glass slide, a few milligrams of Di-I crystals, and a glass pipette with rubber bulb.
Visualizing Di-I-coated tracks in fixed tissue:
The Di-I track will remain visible after fixation and after fluorescent immunocytohistochemistry procedures. We have found that Di-I does not always remain visible after Nissl staining. If you want to use fluorescent methods to label the cell bodies, such as anti-NeuN staining, then no additional procedures are necessary; the Di-I will simply remain after staining. If you want to use Nissl staining or some similar technique, then we recommend photographing the sections after slicing to have a visual record of the electrode track, and then perform staining on these numbered slides. With Nissl staining, it is common for the Di-I to be washed away.
References:
Heimel JA, Van Hooser SD, Nelson SB. Laminar organization of response properties in primary visual cortex of the gray squirrel (Sciurus carolinensis). J Neurophysiol 94:3538-54, 2005. [an example of photographing first and Nissl staining later.]
MSDS: Chloroform
MSDS: Di-I
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