TY - JOUR

T1 - CLARITY-compatible lipophilic dyes for electrode marking and neuronal tracing

AU - Jensen, Kristian HR

AU - Berg, Rune W.

PY - 2016/9/6

Y1 - 2016/9/6

N2 - Fluorescent lipophilic dyes, such as DiI, stain cellular membranes and are used extensively for retrograde/anterograde labeling of neurons as well as for marking the position of extracellular electrodes after electrophysiology. Convenient histological clearing techniques, such as CLARITY, enable immunostaining and imaging of large volumes for 3D-reconstruction. However, such clearing works by removing lipids and, as an unintended consequence, also removes lipophilic dyes. To remedy this wash-out, the molecular structure of the dye can be altered to adhere to both membranes and proteins so the dye remains in the tissue after lipid–clearing. Nevertheless, the capacity of such modified dyes to remain in tissue has not yet been tested. Here, we test dyes with molecular modifications that make them aldehyde-fixable to proteins. We use three Dil–analogue dyes, CM-DiI, SP-DiI and FM 1–43FX that are modified to be CLARITY-compatible candidates. We use the challenging adult, myelin-rich spinal cord tissue, which requires prolonged lipid–clearing, of rats and mice. All three dyes remained in the tissue after lipid–clearing, but CM-DiI had the sharpest and FM 1–43FX the strongest fluorescent signal.

AB - Fluorescent lipophilic dyes, such as DiI, stain cellular membranes and are used extensively for retrograde/anterograde labeling of neurons as well as for marking the position of extracellular electrodes after electrophysiology. Convenient histological clearing techniques, such as CLARITY, enable immunostaining and imaging of large volumes for 3D-reconstruction. However, such clearing works by removing lipids and, as an unintended consequence, also removes lipophilic dyes. To remedy this wash-out, the molecular structure of the dye can be altered to adhere to both membranes and proteins so the dye remains in the tissue after lipid–clearing. Nevertheless, the capacity of such modified dyes to remain in tissue has not yet been tested. Here, we test dyes with molecular modifications that make them aldehyde-fixable to proteins. We use three Dil–analogue dyes, CM-DiI, SP-DiI and FM 1–43FX that are modified to be CLARITY-compatible candidates. We use the challenging adult, myelin-rich spinal cord tissue, which requires prolonged lipid–clearing, of rats and mice. All three dyes remained in the tissue after lipid–clearing, but CM-DiI had the sharpest and FM 1–43FX the strongest fluorescent signal.

KW - Faculty of Health and Medical Sciences

KW - Microscopy

KW - Staining and Labeling

KW - Neuroscience

KW - Electrophysiology

U2 - 10.1038/srep32674

DO - 10.1038/srep32674

M3 - Journal article

C2 - 27597115

VL - 6

JO - Scientific Reports

JF - Scientific Reports

SN - 2045-2322

M1 - 32674

ER -