This worksheet summarizes most of the procedures used as retrograde neuronal tracers using fluorescent latex microspheres or beads. Special information about green beads is at the end of this agreement. Further details are proposed in the following literature: Katz, LC, burkhalter, a. and Dreyer, WD fluorescent latex microspheres are used as retrograde neuromarkers for in vivo and in vitro visual cortex research. Nature 310:498-500 (1984), LC, Katz and DM iarovici, green fluorescent latex microspheres: a new retrograde tracer. Neuroscience 34:511-520 (1990). For questions, questions or comments on the use of this material, please contact 021-50724187 directly.
How to provide: a closed vial contains a concentrated bead solution suspended in distilled water. If red beads are used for retrograde tracing of neuronal pathways, the solution can be used or diluted as it is. In the visual cortex of rats, when red beads were used, dilution with 1:4 did not seem to reduce the quality or degree of retrograde markers. However, for the initial experiment, we strongly recommend the full strength of the solution. In addition to distilled water, the standard salt solution (NaCl, KCl) can also be used as diluent. The green beads provided are fully ready for reverse tracking experiments. Dilution of green beads is not recommended.
Storage: the bead solution shall be stored in humidified containers and in refrigerators to prevent evaporation. Don't freeze! Frozen beads will not work or be saved. If the beads dry, they will not be able to recover. The shelf life of the material has not been determined, but if properly stored, it will remain in good condition for several years.
Application: it is best to use a pressure (e.g. 1 ml) injection beads Hamilton syringe or pressurized air injection system. For local loop operation, a very small volume (30-50 NL) has been injected through a glass pipette with a 30-50 um diameter tip. For conventional retrograde tracing, a larger volume (0.1-0.3 UL) and a larger diameter pipette suction head are used. However, even if the injection volume is large, the beads will not spread far away from the injection position (usually less than 1 mm). Therefore, several injections should be performed to mark all or most of the neurons projected to large structures. It is not recommended to use ion electroosmotic therapy beads as an effective method for the delivery of tracer. But beads do have net negative charges.
Survival time: in most warm blood vertebrate systems, the minimum effective survival time after injection is about 24 hours. The intensity of the markers increased with a longer survival time (up to 48 hours). After 48 hours, no increase (or decrease) in the marking strength was observed. These values may vary greatly in cold-blooded animals, and it is suggested that the initial survival time is one week. The maximum survival time has not been determined, but the quality or extent of the label has not changed even after 14 months of survival. Cells may have been permanently marked. No toxic effects on animals or neurons have been observed.
Fixation and treatment: the standard fixation is to wash with 0.1M phosphate buffer first, and then wash with 4% oligoformaldehyde in 0.1M phosphate buffer (pH 7.4). Glutaraldehyde can produce a large amount of tissue autofluorescence, which may make the bead labeled neurons fuzzy, if possible, should be avoided. Green beads in glutaraldehyde fixation materials will not be visible at all. The frozen sections were collected in phosphate buffer, and installed on the slide covered by gelatin and dried. After completely dry, the slide can be cleaned in xylene for 1 minute and covered with fluoromount or krystalon. Fluorescent lamps can be purchased from atomergic chemicals Corp., New York, famingdale; krystalon, Gibbston, New Jersey, from harleco (EM industries) are briefly exposed to alcohol and xylene, but long exposure (more than 5 minutes) can destroy beads. Beads are very sensitive to glycerin, and if installed in glycerin containing solutions, beads will fade rapidly. Methyl salicylate is superior to glycerin when non permanent cleaning / sealing agent is indicated. If you put the slide in the dark, the label in the cell will not fade for at least one year (although spontaneous fluorescence in the background may increase significantly). So far, attempts to keep the bead label after plastic cladding have not been successful.
Observation: the dye in red beads is rhodamine, so any fluorescent filter for rhodamine can be used. Some older Nikon rhodamine filters have a high background, which may make the labeled cells invisible. Zeiss and Leeds standard rhodamine filters have achieved good results. For green beads, the wide band fluorescent filter has a good effect. Lucifer yellow filters give a stronger signal, but at the cost of higher backgrounds. Narrow band fluorescent filters produce much weaker signals than broadband filters. Even after a long period of observation or micrograph, beads will not fade obviously.
Labels are usually invisible on low power, low numerical aperture dry objects (e.g. x4, X10). If cells are strongly labeled, X10 immersion objective (0.4 or greater digital aperture) or a higher power dry objective usually shows cells. However, X25 immersion lenses often show clearly marked batteries, while low-power objective lenses are lost. These warnings are particularly suitable for green beads. Before determining that the experiment is not working, check the slice near the injection site with immersion objective. There should be many locally labeled cells.
Other information for green bead users: so far, in the work done, it looks like
