| Haber 1998 |
Performed a tracing study with Wheat Germ Agglutinin Horseradish Peroxidase. Two incubation solutions and temperatures were used. The tissue was incubated in a culture medium containing 10% glucose, 10% fetal bovine serum, and 10% penicillin streptomycin or a calcium-low phosphate buffer at either 4°C or 37°C |
Not all cells/axons in a given area possessed labeling Axon and cell body labeling was enhanced under the 4°C conditions Under both the 37°C and 4°C conditions, the calcium-low phosphate buffer had better distance and staining than the culture medium The lower temperature enhanced the number of fibers seen and the distance traced.
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| Kikkawa et al. 2007 |
This study compared three techniques to visualize the cochlear nerve fibers, namely DiI, the modified Golgi-Cox method and NeuroVue dyes. |
For the carbocyanine dyes, DiI, DiO and DiD were used. DiI was suggested to have the best staining among the carbocyanines The problem with the modified Golgi-Cox method was that it does not stain afferent fibers For all the different visualization methods tested, NeuroVue gave the best spatial resolution and fastest staining.
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| Chen et al. 2005 |
Comparison study between DiI/DiO for the tracer, 1%/4% PFA for the preferred concentration, and RT vs. 37°C for the optimal temperature. |
DiI diffused substantially longer distances than DiO. Both dyes migrated farther distances at 37°C. Therefore, higher temperatures facilitate diffusion There was no difference between the tissue tracings incubated in 4% vs 1% PFA. However, 4% PFA was still required for the fixation and post fixation procedure.
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| Lukas et al 1998 |
This study sought to optimize the axon tracing procedure in human and guinea pig tissue. |
Among DiI, DiA and DiO, DiI appeared to be the best tracer. The tissue was incubated at 37°C and 40°C for 3wks-1 year. After 15 weeks, no advancement was seen in the distance traced. Thus, the optimal tracing time was suggested to be 12–15 weeks. It was also suggested there was no difference between incubation at 37°C and 40°C. Since both central nervous system and peripheral nervous system tissue was traced in both guinea pig tissue and human tissue, no difference in tracing quality was seen in the two tissue types. However, a slight difference in distance was observed between the human and guinea pig tissue. Because carbocyanines are compatible with immunohistochemistry techniques, it was suggested Tween 20 was an optimal detergent than Trition X. It was also suggested Trition X may facilitate fading. In regards to sectioning, it was suggested cryostat sections may be less stable than vibratome sections. There was no observable difference between 2% PFA and 4% PFA for the incubation solution.
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| Murphy and Fox 2007 |
This study compared DiI crystals to oil 0.1%, EDTA vs no EDTA, and PBS mounting medium vs glycerol and n-propyl gallate |
When comparing the DiI, they suggested crystals give a better quality and rate of diffusion The researchers suggest long incubation periods promote DiI leakage out of the nerve fiber. It is believed that calcium in the tissue promotes diffusion out of the neuron and promotes unspecific labeling. Therefore, in order to combat this, 0.1% EDTA is suggested to reduce this unspecific labeling. Lastly, PBS is suggested to be a superior mounting solution to glycerol with n-propyl gallate. Glycerol and n-propyl gallate reduced clarity and promoted leakage of the dye which ultimately results in a loss of specific labeling.
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| Hofmann et al 1999 |
The researchers aimed to improve the staining and limit transneuronal labeling. They investigated the effect of increased temperatures and calcium. |
The presence of calcium facilitates dye leakage out of the membranes; therefore, the addition of 0.1% EDTA to the 4% PFA eradicated transneuronal labeling. In regards to temperature, 6°C, room temperature, 30°C, 35°C and 40°C, the researchers found that as the temperatures get higher, the staining becomes progressively more diffuse. It is suggested that increased temperatures accelerate dye diffusion out of the neuron.
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| Fritzsch et al 2005 |
Compared NeuroVue Red, Green, and Maroon with DiI, DiO, DiA and DiD. |
DiO and DiA have slow diffusion in fixed tissue and are not as applicable as a long range tracer. NV dyes were consistently seen to trace longer distances and have the ability to be imaged at lower concentrations than existing dyes. They also diffuse faster than the carbocyanines. The red emitting and near infrared emitting dyes (NV Red, DiI, DiD, NV Maroon) were consistently seen to travel longer distances than the green emitting dyes. NV Maroon appeared to be the easiest to detect over long distances
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