Long-Distance Three-Color Neuronal Tracing in Fixed Tissue Using NeuroVue Dyes

Dissecting development of neuronal connections is critical for understanding neuronal function in both normal and diseased states. Charting the development of the multitude of connections is a monumental task, since a given neuron typically receives hundreds of convergent inputs from other neurons and provides divergent outputs for hundreds of other neurons. Although progress is being made utilizing various mutants and/or genetic constructs expressing fluorescent proteins like GFP, substantial work remains before a database documenting the development and final location of the neuronal pathways in an adult animal is completed. The vast majority of developing neurons cannot be specifically labeled with antibodies and making specific GFP-expressing constructs to tag each of them is an overwhelming task. Fortunately, fluorescent lipophilic dyes have emerged as very useful tools to systematically compare changes in neuronal networks between wild-type and mutant mice. These dyes diffuse laterally along nerve cell membranes in fixed preparations, allowing tracing of the position of a given neuron within the neuronal network in murine mutants fixed at various stages of development. Until recently, however, most evaluations have been limited to one, or at most, two color analyses. We have previously reported three color neuronal profiling using the novel lipophilic dyes NeuroVue (NV) Green, Red and Maroon (Fritzsch et al., Brain. Res. Bull. 66:249–258, 2005). Unfortunately such three color experiments have been limited by the fact that NV Green and its brighter successor, NV Emerald, both exhibit substantially decreased signal intensities when times greater than 48 hours at 37°C are required to achieve neuronal profile filling (unpublished observations). Here we describe a standardized test system developed to allow comparison of candidate dyes and its use to evaluate a series of 488 nm-excited green-emitting lipophilic dyes. The best of these, NV Jade, has spectral properties well matched to NV Red and NV Maroon, better solubility in DMF than DiO or DiA, improved thermostability compared with NV Emerald, and the ability to fill neuronal profiles at rates of 1 mm per day for periods of at least 5 days. Use of NV Jade in combination with NV Red and NV Maroon substantially improves the efficiency of connectional analysis in complex mutants and transgenic models where limited numbers of specimens are available

Cyt‐Geist: Current and Future Challenges in Cytometry: Reports of the CYTO 2019 Conference Workshops

The need for cytometry instrumentation, reagents, training and scientific collaborations in the nations of Africa remains high despite strong efforts by both the African and foreign biomedical and cytometry research communities. Dr. Tesfa and Dr. Blanco therefore organized the first Cytometry in Africa Workshop at CYTO2019. This workshop had several goals. The first goal was to present the results of a pre-workshop survey aimed at assessing flow cytometry resources, personnel, experience and training in Africa. The results of this survey demonstrated important strengths in the African cytometry community, but also pinpointed areas where instrument access, reagent availability and training could be improved. The second goal was to present several collaborative scientific projects in Africa with participation by ISAC members. Third, both existing and proposed strategies for improving collaborative efforts and research support were presented, including cytometer donations, research collaborations and training programs. Finally, an open roundtable discussion was held with workshop attendees, many with experience in working in Africa. A diverse array of investigators from government, academia and industry attended and contributed to the workshop. A key outcome of the workshop was the establishment an African Working group in collaboration with the ISAC Instruments 4 Science Task Force, the ISAC Live Education Task Force, and the ISAC Education Committee. The workshop also marked the establishment of I4S, with the goal of advancing flow cytometry in the international research communit