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. 2019 Aug 19;9(1):12084.
doi: 10.1038/s41598-019-48460-2.

Optimisation and validation of hydrogel-based brain tissue clearing shows uniform expansion across anatomical regions and spatial scales

Adam L Tyson  1   2   3 , Ayesha M Akhtar  4 , Laura C Andreae  5   6
Affiliations

Optimisation and validation of hydrogel-based brain tissue clearing shows uniform expansion across anatomical regions and spatial scales

Adam L Tyson et al. Sci Rep. .

Abstract

Imaging of fixed tissue is routine in experimental neuroscience, but is limited by the depth of tissue that can be imaged using conventional methods. Optical clearing of brain tissue using hydrogel-based methods (e.g. CLARITY) allows imaging of large volumes of tissue and is rapidly becoming commonplace in the field. However, these methods suffer from a lack of standardized protocols and validation of the effect they have upon tissue morphology. We present a simple and reliable protocol for tissue clearing along with a quantitative assessment of the effect of tissue clearing upon morphology. Tissue clearing caused tissue swelling (compared to conventional methods), but this swelling was shown to be similar across spatial scales and the variation was within limits acceptable to the field. The results of many studies rely upon an assumption of uniformity in tissue swelling, and by demonstrating this quantitatively, research using these methods can be interpreted more reliably.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Mouse brain tissue incubated in hydrogel (A4B5P4), cleared using SDS and RI matched using 85% glycerol. (A) Mouse brain prior to tissue clearing, (B) 2 mm section showing the end point of clearing, (C) Cleared whole brain.
Figure 2
Figure 2
Examples of successful antibody stains in mouse brain tissue. Scale bars 100 μm. (A) CTIP2 staining of striatal spiny projection neurons, (B) CUX1 staining of layer II–IV cortical projection neurons, (C) Calbindin staining in olfactory bulb glomerular layer, (D) Parvalbumin staining of primary somatosensory cortical interneurons, (E) Neurofilament staining of striatal white matter tracts, (F) GFAP staining of cerebellar Bergmann glia, (G) NeuN staining in somatosensory cortex, (H) MBP staining of striatal white matter tracts.
Figure 3
Figure 3
Antibody penetration depth as a function of incubation time (equal for primary and secondary antibodies), fitted with an exponential .
Figure 4
Figure 4
Other fluorescent stains in mouse brain tissue. All in primary somatosensory cortex and scale bars 100 μm other than fluromyelin (cortex and striatum scale bar 1 mm). (A) DAPI, (B) Propidium iodide, (C) SYTOX green, (D) SYTOX red, (E) Neurotrace red, (F) Fluromyelin green.
Figure 5
Figure 5
Cell counts of CTIP2- and parvalbumin-positive cells in cortex and striatum, comparison between uncleared and cleared mouse brain tissue. Mean shown as a horizontal line.
Figure 6
Figure 6
Volumes of CTIP2- and parvalbumin-positive cells in cortex and striatum, comparison between uncleared and cleared mouse brain tissue. Mean shown as a horizontal line.
Figure 7
Figure 7
Comparison of cortical thickness in cleared and uncleared tissue, in motor and barrel cortices. Mean shown as a horizontal line.
Figure 8
Figure 8
CVs for each measure in uncleared tissue with the CV due to clearing overlaid.
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