Chemical Clearing and Dehydration of GFP Expressing Mouse Brains

Generally, chemical tissue clearing is performed by a solution consisting of two parts benzyl benzoate and one part benzyl alcohol. However, prolonged exposure to this mixture markedly reduces the fluorescence of GFP expressing specimens, so that one has to compromise between clearing quality and fluorescence preservation. This can be a severe drawback when working with specimens exhibiting low GFP expression rates. Thus, we screened for a substitute and found that dibenzyl ether (phenylmethoxymethylbenzene, CAS 103-50-4) can be applied as a more GFP-friendly clearing medium. Clearing with dibenzyl ether provides improved tissue transparency and strikingly improved fluorescence intensity in GFP expressing mouse brains and other samples as mouse spinal cords, or embryos. Chemical clearing, staining, and embedding of biological samples mostly requires careful foregoing tissue dehydration. The commonly applied tissue dehydration medium is ethanol, which also can markedly impair GFP fluorescence. Screening for a substitute also for ethanol we found that tetrahydrofuran (CAS 109-99-9) is a more GFP-friendly dehydration medium than ethanol, providing better tissue transparency obtained by successive clearing. Combined, tetrahydrofuran and dibenzyl ether allow dehydration and chemical clearing of even delicate samples for UM, confocal microscopy, and other microscopy techniques

Deregulated expression of angiogenesis genes in <i>Prdm6</i>-deficient yolk sacs.

<p>Real-time RT-PCR analysis of selected transcripts identified <i>via</i> global gene expression profiling analysis (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081833#pone.0081833.s003" target="_blank">Fig. S3</a>). The expression values obtained from <i>Prdm6</i>-deficient (del/del) yolk sac samples were normalized to the expression values detected in wild type control samples. The housekeeping gene <i>Tbp</i> was expressed at equivalent levels in wild type and <i>Prdm6</i>-deficient yolk sacs.</p

<i>Prdm6</i> deficiency results in embryonic lethality.

<p>(<b>A</b>) <i>Prdm6</i>^wt/del mice were intercrossed. Pregnant mice were euthanized and embryos dissected and genotyped at defined developmental stages. The percentages of viable embryos of the respective genotypes at the different stages of embryonic development (dpc = days post coitum) are indicated; wild type <i>Prdm6</i>^wt/wt (wt/wt) and heterozygous <i>Prdm6</i>^wt/del (wt/del) mice are viable, whereas <i>Prdm6</i>-deficient <i>Prdm6</i>^{<i>del/del</i>} (del/del) embryos begin to die after E10.0, with no <i>Prdm6</i>^{<i>del/del</i>} embryos being found at developmental stages beyond E16.0. (<b>B</b>) Northern blot analysis of <i>Prdm6</i> expression using total embryonic RNA from different developmental stages from wild type embryos. <i>Gapdh</i> expression analysis served as a loading control. (<b>C</b>) Representative wild type control and <i>Prdm6</i>-deficient embryos (del/del) at the indicated developmental stages. White arrows indicate edematous swelling. (<b>D</b>) Transverse heart sections from wild type control and <i>Prdm6</i>-deficient embryos were stained with H&E and analyzed by microscopy. The thin myocardium of <i>Prdm6</i>-deficient embryos (del/del) is indicated by an arrow. Scale bars correspond to 200 µm. </p

Prdm6 affects angiogenic patterning.

<p>(<b>A</b>) Unusual clusters of densely growing vessel structures on yolk sacs of <i>Prdm6</i>-deficient embryos, as observed under a stereomicroscope. Scale bars correspond to 1 mm (left panel) or 100 µm (right panel). (<b>B</b>) Large vessels in the yolk sacs of E12.5 control and <i>Prdm6</i>-deficient (del/del) embryos under a stereomicroscope. Scale bars correspond to 500 µm. (<b>C</b>) Visualization of E10.5 (left panels) and E11.5 (right panels) yolk sac microvascular systems <i>via</i> immunofluorescent staining with an anti-CD31 primary antibody and a Cy3-conjugated secondary antibody. Scale bars correspond to 200 µm. (<b>D</b>) Quantitative morphometric analysis of the yolk sac vasculature as shown in (C). Avascular space and mean vessel diameters of yolk sacs at E10.5 – E 11.5 are shown as mean ± SEM, n=6. More details about this analysis are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081833#pone.0081833.s002" target="_blank">Figure S2</a>.</p