We demonstrate the use of the gene gun to introduce fluorescent dyes, such as DiI, into neurons in brain slices from rodents and non-human primates of different ages. In this particular case, we use adult mice (3-6 months old) and adult cynomologus monkeys (9-15 years old). This technique, originally described by the laboratory of Dr. Lichtman (Gan et al., 2000), is well suited for the study of dendritic branching and dendritic spine morphology and can be combined with traditional immunostaining, if detergents are kept at a low concentration.
Total time
~4-6 hours per experiment (including slice preparation, labeling, and imaging)
Model organism
Mouse (C57BL/6J or similar, 3-6 months); Cynomolgus monkey (9-15 years)
Steps
1
Prepare DiI and tungsten beads for labeling
Coat tungsten particles with DiI fluorescent dye using organic solvents and allow adequate drying before loading into gene gun cartridges.
▶ 01:56
2
Prepare PVP solution and tubing assembly
Mix polyvinylpyrrolidone solution and assemble gene gun tubing components to optimize ballistic particle delivery pressure and consistency.
▶ 03:24
3
Apply DiI staining to fixed brain slices
Fire the gene gun to deliver DiI-coated beads into prepared brain sections, then rinse and visualize labeled neurons under fluorescence microscopy.
▶ 05:02
4
Perform antibody staining and slide mounting
Apply immunofluorescent antibodies at low detergent concentrations to avoid dye quenching, then mount sections for confocal or epifluorescence imaging.
▶ 06:14
5
Acquire and analyze representative fluorescence images
Image labeled neurons to visualize dendritic branching patterns and spine morphology, demonstrating successful DiOLISTIC labeling across species and age groups.
▶ 07:39