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In vivo imaging of oligodendrocytes with sulforhodamine 101

Nature Methods volume 11, pages 1081–1082 (2014)Cite this article

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The fluorescent dye sulforhodamine 101 (SR101) has been widely used for in vivo brain imaging because of its reported ability to exclusively label astrocytes, despite a note of caution in the original publication1. After administration, the dye diffuses through the astrocyte syncytium via gap junctions, brightly labeling astrocyte cell bodies, processes and perivascular endfeet. SR101 has been used extensively in combination with calcium-sensitive dyes to distinguish calcium signals derived from neurons and astrocytes2,3.

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Figure 1: SR101 labels both astrocytes and myelinating oligodendrocytes in vivo.

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Acknowledgements

This study was supported by grants from the US National Institutes of Health (R01HL106815 and R21NS0887511) and the US National Multiple Sclerosis Society (PP2154) to J.G. We thank A. Nishiyama (University of Connecticut) for sharing NG2cre:ZEG transgenic mice. We thank F. Vaccarino (Yale University) for sharing Aldh1L1-GFP transgenic mice.

Author information

Authors and Affiliations

  1. Department of Neurology, Yale School of Medicine, New Haven, Connecticut, USA

    Robert A Hill & Jaime Grutzendler

  2. Department of Neurobiology, Yale School of Medicine, New Haven, Connecticut, USA

    Robert A Hill & Jaime Grutzendler

Authors
  1. Robert A Hill
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  2. Jaime Grutzendler
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Contributions

R.A.H. and J.G. conceived of and designed the study. R.A.H. performed the experiments. R.A.H and J.G. analyzed the data and wrote the paper.

Corresponding author

Correspondence to Jaime Grutzendler.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 SR101 does not label neurons, microglia, NG2 cells, or vascular pericytes.

a) In vivo two photon fluorescence image captured from the cortex of a Thy1-YFP transgenic mouse demonstrating that YFP (green) labeled neuronal cell bodies (arrowheads) and dendrites are not labeled with SR101 (red, arrows). scale bars 25μm, 10μm. b) In vivo image captured from the cortex of a CX3CR1-GFP transgenic mouse demonstrating that GFP labeled microglia (green, arrowheads) are not labeled with SR101 (red, arrows), scale bars 25μm, 10μm. c) In vivo image captured from the cortex of an NG2cre:ZEG transgenic mouse demonstrating that GFP labeled NG2 cells and vascular pericytes (green, arrowheads) are not labeled with SR101 (red, arrows), scale bars 25μm, 10μm. Images representative of data acquired from 3 mice for each genotype.

Supplementary Figure 2 Morphological properties of single SR101 -labeled cells.

a) Low magnification in vivo image showing SR101 (red) labeling in an Aldh1L1-GFP (green) transgenic mouse with blood vessels imaged via intravascular injection of a high molecular weight dextran (cyan), scale bar 50μm. b) In vivo image showing a single astrocyte (a) and a single oligodendrocyte (o) both labeled with SR101 (red) but only the astrocyte showing the typical morphology with perivascular endfeet (arrows), scale bar 20μm. c) Multiple examples of single astrocytes labeled with SR101 with perivascular endfeet (arrows), scale bars 10μm. Images representative of data acquired from 3 mice and quantification can be found in the main text.

Supplementary Figure 3 SR101 labels oligodendrocyte cell bodies and myelinating processes.

a) In vivo image showing a single GFP (green) labeled oligodendrocyte from an NG2cre:ZEG transgenic mouse showing SR101 (red) labeling of proximal non-myelinating processes (arrows) and SCoRe (cyan) labeled myelinating processes (arrowheads). * depicts a node of Ranvier, scale bars 10μm, 5μm. b) In vivo image from a PLPcreER:mT/mG transgenic mouse showing SR101 (red) labeling in proximal non-myelinating processes (arrow) and in membrane tethered GFP-labeled (green), SCoRe-labeled (cyan), myelinating processes (arrowheads) scale bar 10μm.

Supplementary Figure 4 Developmental density and identity of SR101 -labeled cells.

a) Representative in vivo two photon fluorescence images captured from cortex of NG2cre:ZEG mice labeled with SR101 at P30 and P90 demonstrating the differences in the density of GFP (green) SR101 (red) labeled oligodendrocytes, scale bars 50μm. b) Quantification showing the total density of SR101 labeled cells (left), cells identified as astrocytes (middle) and the cells identified as oligodendrocytes (right) at postnatal (P) days 30, 90 and 210 by cortical depth, error bars = s.d. * P < 0.05 compared to P30, * P < 0.05 compared to P90, student's t-test, data acquired from 3 mice at each age.

Supplementary Figure 5 Intravenous injection of SR101 labels oligodendrocytes and astrocytes in vivo.

a) In vivo images captured through a thinned skull 24 hours after intravenous injection of SR101 in NG2cre:ZEG transgenic mice. Arrows indicate labeling of mature oligodendrocytes with SR101 (red) and GFP (green) while other GFP labeled cells (NG2 cells and vascular pericytes) are not labeled, consistent with topical application of SR101 to an open skull preparation. Scale bars 20μm. Images representative of data acquired from 3 mice.

Supplementary Figure 6 Temporal dynamics of cell-specific labeling with SR101 in NG2cre:ZEG mice.

a) In vivo time lapse images of a GFP+ (green, arrow) oligodendrocyte in an NG2cre:ZEG mouse showing SR101 (red) labeling over time (indicated in minutes), scale bars 5μm. b) Images taken at 40 and 140 minutes after SR101 labeling indicating dye spread from astrocytes to oligodendrocytes (arrows) in NG2cre:ZEG mice, scale bar 50μm. c) Quantification showing significant differences in the change in fluorescent intensity starting at 40 min after SR101 labeling , error bars = s.e.m. *P < 0.05, t-test, data acquired from 3 mice.

Supplementary Figure 7 Temporal dynamics of cell-specific labeling with SR101 in Aldh1L1-GFP mice.

a) In vivo time lapse images showing SR101(red) labeling over time of a GFP- (arrow) cell in an Aldh1L1-GFP mouse, scale bar 5μm. b) Representative images taken at 40 and 140 minutes after SR101 labeling indicating spread of the dye from astrocytes to oligodendrocytes (arrows), scale bar 50μm. Images representative of data acquired from 3 mice.

Supplementary Figure 8 Temporal dynamics of cell-specific labeling with SR101 in PLPcreER:mT/mG mice.

a) In vivo image captured from the cortex of a PLPcreER:mT/mG transgenic mouse demonstrating that membrane bound GFP (green) labeled myelinating oligodendrocytes (arrowheads) are not initially labeled with SR101 (red) but become labeled between 30 and 120 minutes after SR101 application, scale bars 25μm. Images representative of data acquired from 3 mice.

Supplementary Figure 9 Carbenoxolone inhibits SR101 labeling of both oligodendrocytes and astrocytes.

a) Timeline of the experimental procedure for blocking SR101 labeling in vivo with the gap junction blocker carbenoxolone (CBX, 100μM). b) In vivo images from NG2cre:ZEG (green) mice showing SR101 (red) labeling in control conditions and after CBX treatment, scale bar 25μm. c) Fluorescence intensity quantification of SR101 labeling normalized to GFP intensity showing significant inhibition of SR101 labeling under CBX treatment in both astrocytes and oligodendrocytes, error bars = s.d. *P < 0.002, student's ttest, data acquired from 3 mice for each treatment.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–9 and Supplementary Methods (PDF 1657 kb)

Supplementary Video 1

In vivo SR101 labelling in Aldh1L1-GFP mice (AVI 2492 kb)

Supplementary Video 2

In vivo SR101 labelling in NG2cre:ZEG mice (AVI 2487 kb)

Supplementary Video 3

Temporal profile of cell specific labelling with SR101 in NG2cre:ZEG mice (AVI 364 kb)

Supplementary Video 4

Temporal profile of cell specific labelling with SR101 in Aldh1L1-GFP mice (AVI 526 kb)

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Hill, R., Grutzendler, J. In vivo imaging of oligodendrocytes with sulforhodamine 101. Nat Methods 11, 1081–1082 (2014). https://doi.org/10.1038/nmeth.3140

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