Biology Reference
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22. If stringy precipitate is seen floating, it may be DNA released from dead cells.
Remove it by adding DNase ( see Subheading 2.3.3. for stock solution) to
0.1-0.2 mg/mL final after tripling the volume with complete medium to dilute
the dye. Continue the incubation for 5-15 min, until precipitate dissolves.
23. 4% BSA is more dense than BSS. The cushion efficiently separates the cells from
dye, enzymes, and debris left from the labeling and dissociation.
24. We routinely plated 1-2
10 6
L on parafilm. This gives
1000-2000 cells/mm 2 in the middle portion of the disk. At the edges, densities
are lower due to the shape of the medium bubble on the disk.
×
cells/disk in 90
µ
25.
This step can be done immediately after the slices are cut or after a period of slice
preculture and/or treatment. We routinely plated dissociated cells 4-5 h after slicing.
26.
Transfer of cocultures through an air/liquid interface will remove cells that are
not well attached to the slice.
27.
After fixation, the cocultures can better withstand manipulation through an air/
liquid interface.
28.
If immunostaining is performed, bisbenzamide can be added after this procedure,
before mounting the slides. Rinsing with PBS after the bisbenzamide may slightly
lower the background fluorescence, however, the stain is effective when slices
are mounted directly from the dye solution.
29.
Cocultures grown in well inserts can also be mounted this way by cutting the mem-
brane out of the insert with a scalpel blade and mounting the membrane like a disk.
Acknowledgments
This work was supported by NIH grant NS26862.
References
1. Emerling, D. E. and Lander, A. D. (1994) Laminar specific attachment and neurite
outgrowth of thalamic neurons on cultured slices of developing cerebral neocor-
tex. Development 120, 2811-2822.
2. Emerling, D. E. and Lander, A. D. (1996) Inhibitors and promoters of thalamic
neuron adhesion and outgrowth in embryonic neocortex: functional association
with chondroitin sulfate. Neuron 17, 1089-1100.
3. Honig, M. G. and Hume, R. I. (1986) Fluorescent carbocyanine dyes allow living
neurons of identified origin to be studied in long-term cultures. J. Cell Biol. 103,
171-187.
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