Chemical structures of tetrahydrofuran (THF) and dibenzyl ether (DBE).

<p><b>A</b>: THF. <b>B</b>: DBE. Interestingly, both compounds are ethers without further functional groups.</p

UM-reconstructions of isolated hippocampi of a thy-1 EGFP-M mouse.

<p>DBE clearing provides improved fluorescence and visibility of details. <b>A</b>: left hippocampus dehydrated with THF and cleared with BABB. <b>B</b>: corresponding right hippocampus dehydrated with THF and cleared with DBE. <b>A1–B1</b>: Olympus objective XLFluar 4×, N.A. 0.28, scale bar 100 µm. (<b>A2–B2</b>) 4× Olympus objective XLFluar 4×, N.A. 0.28, scale bar 20 µm. <b>A3–B3</b>: 4× Olympus objective XLFluar 4×, N.A. 0.28, scale bar 10 µm. <b>CA1</b>: cornu ammonis region one. <b>gl</b>: granular cell layer <b>py</b>: pyramidal cells.</p

Removal of peroxides from THF and DBE.

<p><b>A</b>: Apparatus for peroxide cleaning of THF <b>1</b>: Dropping funnel with pressure compensation. <b>2</b>: Chromatography column filled with basic activated aluminum oxide activity grade Brockman 1 <b>3</b>: Two necked round bottom flask <b>4</b>: Drying tube filled with calcium chloride. <b>B</b>: Apparatus for peroxide removal in DBE and BABB <b>1</b>: Filter unit with filter plate (16–40 µm pore size) <b>2</b>: Vacuum tight filtering flask.</p

Quantification of fluorescence preservation during repetitive long-term illumination.

<p>Experiments were performed on three control and three resin-embedded samples. Each sample was exposed for 120 min on 3 consecutive days. Images were recorded any 6 min. On these images, mean pixel intensities were calculated in ROIs either covering areas exclusively containing fluorescence signal or background fluorescence. Size and location of the ROIs, were defined using the first image of each illumination interval. Length of scale bar: 300 µm. (<b>B1–B6</b>) Plots of the relative fluorescence intensity over time. Change of signal and background fluorescence intensities during three successive light exposures (day1 – day3) of 120 min duration each. Photobleaching is much lower in the resin-embedded sample than in the control and limited to the initial illumination phase. During light exposure signal to background ratios (SBRs  =  mean signal intensity/mean background intensity) remain constant for the resin embedded samples (r₁, r₂, r₃), while they exponentially decrease in the controls (c₁, c₂, c₃). The error bars indicate the standard error of the mean (SEM).</p

GFP signal preservation in resin-embedded mouse brain hemispheres.

<p>UM-images of a selected plane within the hippocampal area in both, a control (<b>A</b>) and a resin-embedded brain hemisphere (<b>B</b>), each recorded at onset (<b>1</b>, <b>3</b>, <b>5</b>) and after 120 min of constant high power illumination (<b>2</b>, <b>4</b>, <b>6</b>). (<b>A</b>) The control hemisphere shows a pronounced quenching of GFP-fluorescence during the illumination intervals. (<b>B</b>) In the resin-embedded hemisphere photobleaching is markedly reduced. Although a certain amount of photobleaching occurs during the first illumination interval (B2 vs. B1), the fluorescence intensity remains approximately stable during further light exposures. Note the recovery of fluorescence between illuminations. Length of scale bars: 150 µm.</p

Fabrication of molds for resin embedding from Silastic E-RTV silicone rubber.

<p>(<b>A</b>) Casting frame made from acrylic glass. (<b>B</b>) Silicon rubber mold. (<b>C</b>) Cured resin block with embedded cleared mouse brain hemisphere.</p

The refractive index of the cured resin depends on the relative amount of D.E.R.736 in the resin compound mixture (D.E.R.736 + D.E.R.332).

<p>By varying the percentage of D.E.R.736 the refractive index can be adjusted to different clearing reagents as DBE or BABB. The curing agent IPDA should be added 1:5 (vol/vol) to the premixed resin compound mixture.</p

Tested resin compounds and curing agents.

<p>The table gives an overview on the resins and curing compounds tested to achieve a transparent solid resin block preserving transparency and fluorescence of cleared samples.</p><p>Tested resin compounds and curing agents.</p

Fluorescence preservation by resin embedding during long-term storage.

<p>No loss of GFP signal quality in a resin-embedded mouse hippocampus can be observed within two months. In contrast, a hippocampus stored in DBE loses its fluorescence within less than two weeks. Length of scale bar: 150 µm.</p