Lumafuor's beads were packed in a sealed vial and the concentrated beads were suspended in distilled water. If red beads are used as the reverse tracing of neural pathway, the dilution method is recommended: 1:4 dilution can be used in rat visual cortex without reducing the intensity and quality of fluorescence labeling of beads. However, it is not recommended to use diluted beads for injection tracing. In addition to distilled water, conventional salt solutions such as NaCl and KCl can also be used as diluents. If green beads is used, it is strongly recommended to use the stock solution without dilution.
Harleco (EM industries, Gibbstown, NJ). Slices can only be exposed to ethanol or xylene for a short time, but long-term exposure (more than 5 minutes) can damage beads. Beads is very sensitive to glycerol, and fluorescence will be quickly extracted in glycerin environment. Therefore, glycerin sealing agent should not be used. If it is unavoidable, methyl salicylate can be used instead of glycerin as sealing agent. If the slices were stored in dark environment, the cells labeled with fluorescent beads could not be extracted for one year (but the fluorescence background of the slices would increase). So far, there is no record of beads labeled tissue coated with plastic materials.
It is easy to use pressure injection, such as 1 ml Hamilton microinjector or pneumatic injection system. If small area microinjection (30-50 NL) is needed, glass electrode with 30-50 um diameter at the end can be used for injection, while larger diameter glass electrode can be used for conventional reverse tracing (injection volume 0.1-0.3 UL). However, even at a higher dose, beads does not diffuse significantly from the injection site (usually less than 1 mm). Therefore, in order to label neurons projecting to a larger nucleus as completely as possible, multi-point injection is needed. Although beads are negatively charged, ion permeation method is not recommended for tracing beads.
