Lumafluor’s RetroBeads™ are the original microsphere products developed for retrograde tracing studies and remain the only experimentally validated microsphere system in this field. The green and red fluorescent RetroBeads™ are currently supplied exclusively by Lumafluor.

Lumafluor Red RetroBeads™ are fluorescent microspheres specifically designed for applications in flow cytometry. The product consists of polystyrene polymer microspheres embedded with red fluorescent dye. Each bead has an approximate diameter of 5.5 µm and is supplied as an aqueous suspension.

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• Product Specifications:

The product is supplied as a concentrated aqueous suspension. Red microspheres can be used directly or diluted for neuronal pathway retrograde tracing. In experiments involving the rat visual cortex, a 1:4 dilution still maintained effective labeling; however, the undiluted stock solution is strongly recommended for first-time use. In addition to distilled water, standard saline solutions (e.g., NaCl or KCl) may also be used as diluents. Green microspheres are typically used without dilution.

• Standard Procedure:

Tissue preparation typically follows this sequence: 0.1 M phosphate buffer rinse → fixation with 4% paraformaldehyde in phosphate buffer (pH 7.4). The use of glutaraldehyde should be avoided due to its induction of tissue autofluorescence; green beads are completely invisible in glutaraldehyde-fixed samples.

For cryosections, gelatin-coated slides are recommended. After air drying, clear sections in xylene for approximately 1 minute, and mount using Fluoromount or Krystalon.

Note: Prolonged exposure (>5 minutes) to alcohol or xylene may damage the microspheres. Glycerol-based mounting media may cause fluorescence quenching.

• Key Advantages of Lumafluor RetroBeads™:

1.Highly localized injection sites, ideal for fine-scale connectomics studies

2.Long-term intracellular persistence (>1 year) without detectable toxicity

3.Compatible with most anterograde tracers, in situ hybridization, and immunohistochemistry techniques

• Special Features of Retrobeads IX:

1.Capable of carrying neurotrophic factors, neurotransmitters, agonists, or antagonists to targeted regions

2.Enables precise retrograde identification of neurons exposed to bioactive compounds

3.Special surface treatment significantly enhances adsorption of proteins and other biologically active molecules

4.Demonstrates superior tracing efficiency in primate systems compared with standard beads

• Injection Method:

A pressure injection system is recommended (e.g., 1 mL Hamilton syringe or pneumatic injection system).

1.For local circuit studies: use microinjections of 30–50 nL with glass micropipettes (tip diameter 30–50 μm)

2.For standard retrograde tracing: injection volumes of 0.1–0.3 μL are typically used, requiring a larger needle diameter

Note: The diffusion radius of the beads at the injection site is usually less than 1 mm. Therefore, multiple injection sites may be required to label larger anatomical regions. Iontophoretic injection is not recommended, as the beads carry a negative charge.

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