Abstract

The ability to acquire high resolution 3D images of the heart enables to study heart diseases more in detail. In this work, the CUBIC (clear, unobstructed brain imaging cocktails and computational analysis) clearing protocol was optimized for thick mouse heart sections to enhance the penetration depth of the confocal microscope lasers into the tissue. In addition, the optimized CUBIC clearing of the heart enhances antibody penetration into the tissue by a factor of five. The present protocol enables deep 3D high-quality image acquisition in the heart allowing a much more accurate assessment of the cellular and structural changes that underlie heart diseases.

© 2016 Optical Society of America

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References

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  1. A. Pasipoularides, “Mechanotransduction Mechanisms for Intraventricular Diastolic Vortex Forces and Myocardial Deformations: Part 1,” J. Cardiovasc. Transl. Res. 8(1), 76–87 (2015).
    [Crossref] [PubMed]
  2. A. Pasipoularides, “Mechanotransduction Mechanisms for Intraventricular Diastolic Vortex Forces and Myocardial Deformations: Part 2,” J. Cardiovasc. Transl. Res. 8(5), 293–318 (2015).
    [Crossref] [PubMed]
  3. A. J. Moy, P. C. Lo, and B. Choi, “High-resolution visualization of mouse cardiac microvasculature using optical histology,” Biomed. Opt. Express 5(1), 69–77 (2014).
    [Crossref] [PubMed]
  4. M. Sivaguru, G. Fried, B. S. Sivaguru, V. A. Sivaguru, X. Lu, K. H. Choi, M. T. Saif, B. Lin, and S. Sadayappan, “Cardiac muscle organization revealed in 3-D by imaging whole-mount mouse hearts using two-photon fluorescence and confocal microscopy,” Biotechniques 59(5), 295–308 (2015).
    [Crossref] [PubMed]
  5. D. S. Richardson and J. W. Lichtman, “Clarifying Tissue Clearing,” Cell 162(2), 246–257 (2015).
    [Crossref] [PubMed]
  6. M. D. Muzumdar, B. Tasic, K. Miyamichi, L. Li, and L. Luo, “A global double-fluorescent Cre reporter mouse,” Genesis 45(9), 593–605 (2007).
    [Crossref] [PubMed]
  7. E. A. Susaki, K. Tainaka, D. Perrin, F. Kishino, T. Tawara, T. M. Watanabe, C. Yokoyama, H. Onoe, M. Eguchi, S. Yamaguchi, T. Abe, H. Kiyonari, Y. Shimizu, A. Miyawaki, H. Yokota, and H. R. Ueda, “Whole-Brain Imaging with Single-Cell Resolution Using Chemical Cocktails and Computational Analysis,” Cell 157(3), 726–739 (2014).
    [Crossref] [PubMed]
  8. E. A. Susaki, K. Tainaka, D. Perrin, H. Yukinaga, A. Kuno, and H. R. Ueda, “Advanced CUBIC protocols for whole-brain and whole-body clearing and imaging,” Nat. Protoc. 10(11), 1709–1727 (2015).
    [Crossref] [PubMed]

2015 (5)

A. Pasipoularides, “Mechanotransduction Mechanisms for Intraventricular Diastolic Vortex Forces and Myocardial Deformations: Part 1,” J. Cardiovasc. Transl. Res. 8(1), 76–87 (2015).
[Crossref] [PubMed]

A. Pasipoularides, “Mechanotransduction Mechanisms for Intraventricular Diastolic Vortex Forces and Myocardial Deformations: Part 2,” J. Cardiovasc. Transl. Res. 8(5), 293–318 (2015).
[Crossref] [PubMed]

M. Sivaguru, G. Fried, B. S. Sivaguru, V. A. Sivaguru, X. Lu, K. H. Choi, M. T. Saif, B. Lin, and S. Sadayappan, “Cardiac muscle organization revealed in 3-D by imaging whole-mount mouse hearts using two-photon fluorescence and confocal microscopy,” Biotechniques 59(5), 295–308 (2015).
[Crossref] [PubMed]

D. S. Richardson and J. W. Lichtman, “Clarifying Tissue Clearing,” Cell 162(2), 246–257 (2015).
[Crossref] [PubMed]

E. A. Susaki, K. Tainaka, D. Perrin, H. Yukinaga, A. Kuno, and H. R. Ueda, “Advanced CUBIC protocols for whole-brain and whole-body clearing and imaging,” Nat. Protoc. 10(11), 1709–1727 (2015).
[Crossref] [PubMed]

2014 (2)

E. A. Susaki, K. Tainaka, D. Perrin, F. Kishino, T. Tawara, T. M. Watanabe, C. Yokoyama, H. Onoe, M. Eguchi, S. Yamaguchi, T. Abe, H. Kiyonari, Y. Shimizu, A. Miyawaki, H. Yokota, and H. R. Ueda, “Whole-Brain Imaging with Single-Cell Resolution Using Chemical Cocktails and Computational Analysis,” Cell 157(3), 726–739 (2014).
[Crossref] [PubMed]

A. J. Moy, P. C. Lo, and B. Choi, “High-resolution visualization of mouse cardiac microvasculature using optical histology,” Biomed. Opt. Express 5(1), 69–77 (2014).
[Crossref] [PubMed]

2007 (1)

M. D. Muzumdar, B. Tasic, K. Miyamichi, L. Li, and L. Luo, “A global double-fluorescent Cre reporter mouse,” Genesis 45(9), 593–605 (2007).
[Crossref] [PubMed]

Abe, T.

E. A. Susaki, K. Tainaka, D. Perrin, F. Kishino, T. Tawara, T. M. Watanabe, C. Yokoyama, H. Onoe, M. Eguchi, S. Yamaguchi, T. Abe, H. Kiyonari, Y. Shimizu, A. Miyawaki, H. Yokota, and H. R. Ueda, “Whole-Brain Imaging with Single-Cell Resolution Using Chemical Cocktails and Computational Analysis,” Cell 157(3), 726–739 (2014).
[Crossref] [PubMed]

Choi, B.

Choi, K. H.

M. Sivaguru, G. Fried, B. S. Sivaguru, V. A. Sivaguru, X. Lu, K. H. Choi, M. T. Saif, B. Lin, and S. Sadayappan, “Cardiac muscle organization revealed in 3-D by imaging whole-mount mouse hearts using two-photon fluorescence and confocal microscopy,” Biotechniques 59(5), 295–308 (2015).
[Crossref] [PubMed]

Eguchi, M.

E. A. Susaki, K. Tainaka, D. Perrin, F. Kishino, T. Tawara, T. M. Watanabe, C. Yokoyama, H. Onoe, M. Eguchi, S. Yamaguchi, T. Abe, H. Kiyonari, Y. Shimizu, A. Miyawaki, H. Yokota, and H. R. Ueda, “Whole-Brain Imaging with Single-Cell Resolution Using Chemical Cocktails and Computational Analysis,” Cell 157(3), 726–739 (2014).
[Crossref] [PubMed]

Fried, G.

M. Sivaguru, G. Fried, B. S. Sivaguru, V. A. Sivaguru, X. Lu, K. H. Choi, M. T. Saif, B. Lin, and S. Sadayappan, “Cardiac muscle organization revealed in 3-D by imaging whole-mount mouse hearts using two-photon fluorescence and confocal microscopy,” Biotechniques 59(5), 295–308 (2015).
[Crossref] [PubMed]

Kishino, F.

E. A. Susaki, K. Tainaka, D. Perrin, F. Kishino, T. Tawara, T. M. Watanabe, C. Yokoyama, H. Onoe, M. Eguchi, S. Yamaguchi, T. Abe, H. Kiyonari, Y. Shimizu, A. Miyawaki, H. Yokota, and H. R. Ueda, “Whole-Brain Imaging with Single-Cell Resolution Using Chemical Cocktails and Computational Analysis,” Cell 157(3), 726–739 (2014).
[Crossref] [PubMed]

Kiyonari, H.

E. A. Susaki, K. Tainaka, D. Perrin, F. Kishino, T. Tawara, T. M. Watanabe, C. Yokoyama, H. Onoe, M. Eguchi, S. Yamaguchi, T. Abe, H. Kiyonari, Y. Shimizu, A. Miyawaki, H. Yokota, and H. R. Ueda, “Whole-Brain Imaging with Single-Cell Resolution Using Chemical Cocktails and Computational Analysis,” Cell 157(3), 726–739 (2014).
[Crossref] [PubMed]

Kuno, A.

E. A. Susaki, K. Tainaka, D. Perrin, H. Yukinaga, A. Kuno, and H. R. Ueda, “Advanced CUBIC protocols for whole-brain and whole-body clearing and imaging,” Nat. Protoc. 10(11), 1709–1727 (2015).
[Crossref] [PubMed]

Li, L.

M. D. Muzumdar, B. Tasic, K. Miyamichi, L. Li, and L. Luo, “A global double-fluorescent Cre reporter mouse,” Genesis 45(9), 593–605 (2007).
[Crossref] [PubMed]

Lichtman, J. W.

D. S. Richardson and J. W. Lichtman, “Clarifying Tissue Clearing,” Cell 162(2), 246–257 (2015).
[Crossref] [PubMed]

Lin, B.

M. Sivaguru, G. Fried, B. S. Sivaguru, V. A. Sivaguru, X. Lu, K. H. Choi, M. T. Saif, B. Lin, and S. Sadayappan, “Cardiac muscle organization revealed in 3-D by imaging whole-mount mouse hearts using two-photon fluorescence and confocal microscopy,” Biotechniques 59(5), 295–308 (2015).
[Crossref] [PubMed]

Lo, P. C.

Lu, X.

M. Sivaguru, G. Fried, B. S. Sivaguru, V. A. Sivaguru, X. Lu, K. H. Choi, M. T. Saif, B. Lin, and S. Sadayappan, “Cardiac muscle organization revealed in 3-D by imaging whole-mount mouse hearts using two-photon fluorescence and confocal microscopy,” Biotechniques 59(5), 295–308 (2015).
[Crossref] [PubMed]

Luo, L.

M. D. Muzumdar, B. Tasic, K. Miyamichi, L. Li, and L. Luo, “A global double-fluorescent Cre reporter mouse,” Genesis 45(9), 593–605 (2007).
[Crossref] [PubMed]

Miyamichi, K.

M. D. Muzumdar, B. Tasic, K. Miyamichi, L. Li, and L. Luo, “A global double-fluorescent Cre reporter mouse,” Genesis 45(9), 593–605 (2007).
[Crossref] [PubMed]

Miyawaki, A.

E. A. Susaki, K. Tainaka, D. Perrin, F. Kishino, T. Tawara, T. M. Watanabe, C. Yokoyama, H. Onoe, M. Eguchi, S. Yamaguchi, T. Abe, H. Kiyonari, Y. Shimizu, A. Miyawaki, H. Yokota, and H. R. Ueda, “Whole-Brain Imaging with Single-Cell Resolution Using Chemical Cocktails and Computational Analysis,” Cell 157(3), 726–739 (2014).
[Crossref] [PubMed]

Moy, A. J.

Muzumdar, M. D.

M. D. Muzumdar, B. Tasic, K. Miyamichi, L. Li, and L. Luo, “A global double-fluorescent Cre reporter mouse,” Genesis 45(9), 593–605 (2007).
[Crossref] [PubMed]

Onoe, H.

E. A. Susaki, K. Tainaka, D. Perrin, F. Kishino, T. Tawara, T. M. Watanabe, C. Yokoyama, H. Onoe, M. Eguchi, S. Yamaguchi, T. Abe, H. Kiyonari, Y. Shimizu, A. Miyawaki, H. Yokota, and H. R. Ueda, “Whole-Brain Imaging with Single-Cell Resolution Using Chemical Cocktails and Computational Analysis,” Cell 157(3), 726–739 (2014).
[Crossref] [PubMed]

Pasipoularides, A.

A. Pasipoularides, “Mechanotransduction Mechanisms for Intraventricular Diastolic Vortex Forces and Myocardial Deformations: Part 1,” J. Cardiovasc. Transl. Res. 8(1), 76–87 (2015).
[Crossref] [PubMed]

A. Pasipoularides, “Mechanotransduction Mechanisms for Intraventricular Diastolic Vortex Forces and Myocardial Deformations: Part 2,” J. Cardiovasc. Transl. Res. 8(5), 293–318 (2015).
[Crossref] [PubMed]

Perrin, D.

E. A. Susaki, K. Tainaka, D. Perrin, H. Yukinaga, A. Kuno, and H. R. Ueda, “Advanced CUBIC protocols for whole-brain and whole-body clearing and imaging,” Nat. Protoc. 10(11), 1709–1727 (2015).
[Crossref] [PubMed]

E. A. Susaki, K. Tainaka, D. Perrin, F. Kishino, T. Tawara, T. M. Watanabe, C. Yokoyama, H. Onoe, M. Eguchi, S. Yamaguchi, T. Abe, H. Kiyonari, Y. Shimizu, A. Miyawaki, H. Yokota, and H. R. Ueda, “Whole-Brain Imaging with Single-Cell Resolution Using Chemical Cocktails and Computational Analysis,” Cell 157(3), 726–739 (2014).
[Crossref] [PubMed]

Richardson, D. S.

D. S. Richardson and J. W. Lichtman, “Clarifying Tissue Clearing,” Cell 162(2), 246–257 (2015).
[Crossref] [PubMed]

Sadayappan, S.

M. Sivaguru, G. Fried, B. S. Sivaguru, V. A. Sivaguru, X. Lu, K. H. Choi, M. T. Saif, B. Lin, and S. Sadayappan, “Cardiac muscle organization revealed in 3-D by imaging whole-mount mouse hearts using two-photon fluorescence and confocal microscopy,” Biotechniques 59(5), 295–308 (2015).
[Crossref] [PubMed]

Saif, M. T.

M. Sivaguru, G. Fried, B. S. Sivaguru, V. A. Sivaguru, X. Lu, K. H. Choi, M. T. Saif, B. Lin, and S. Sadayappan, “Cardiac muscle organization revealed in 3-D by imaging whole-mount mouse hearts using two-photon fluorescence and confocal microscopy,” Biotechniques 59(5), 295–308 (2015).
[Crossref] [PubMed]

Shimizu, Y.

E. A. Susaki, K. Tainaka, D. Perrin, F. Kishino, T. Tawara, T. M. Watanabe, C. Yokoyama, H. Onoe, M. Eguchi, S. Yamaguchi, T. Abe, H. Kiyonari, Y. Shimizu, A. Miyawaki, H. Yokota, and H. R. Ueda, “Whole-Brain Imaging with Single-Cell Resolution Using Chemical Cocktails and Computational Analysis,” Cell 157(3), 726–739 (2014).
[Crossref] [PubMed]

Sivaguru, B. S.

M. Sivaguru, G. Fried, B. S. Sivaguru, V. A. Sivaguru, X. Lu, K. H. Choi, M. T. Saif, B. Lin, and S. Sadayappan, “Cardiac muscle organization revealed in 3-D by imaging whole-mount mouse hearts using two-photon fluorescence and confocal microscopy,” Biotechniques 59(5), 295–308 (2015).
[Crossref] [PubMed]

Sivaguru, M.

M. Sivaguru, G. Fried, B. S. Sivaguru, V. A. Sivaguru, X. Lu, K. H. Choi, M. T. Saif, B. Lin, and S. Sadayappan, “Cardiac muscle organization revealed in 3-D by imaging whole-mount mouse hearts using two-photon fluorescence and confocal microscopy,” Biotechniques 59(5), 295–308 (2015).
[Crossref] [PubMed]

Sivaguru, V. A.

M. Sivaguru, G. Fried, B. S. Sivaguru, V. A. Sivaguru, X. Lu, K. H. Choi, M. T. Saif, B. Lin, and S. Sadayappan, “Cardiac muscle organization revealed in 3-D by imaging whole-mount mouse hearts using two-photon fluorescence and confocal microscopy,” Biotechniques 59(5), 295–308 (2015).
[Crossref] [PubMed]

Susaki, E. A.

E. A. Susaki, K. Tainaka, D. Perrin, H. Yukinaga, A. Kuno, and H. R. Ueda, “Advanced CUBIC protocols for whole-brain and whole-body clearing and imaging,” Nat. Protoc. 10(11), 1709–1727 (2015).
[Crossref] [PubMed]

E. A. Susaki, K. Tainaka, D. Perrin, F. Kishino, T. Tawara, T. M. Watanabe, C. Yokoyama, H. Onoe, M. Eguchi, S. Yamaguchi, T. Abe, H. Kiyonari, Y. Shimizu, A. Miyawaki, H. Yokota, and H. R. Ueda, “Whole-Brain Imaging with Single-Cell Resolution Using Chemical Cocktails and Computational Analysis,” Cell 157(3), 726–739 (2014).
[Crossref] [PubMed]

Tainaka, K.

E. A. Susaki, K. Tainaka, D. Perrin, H. Yukinaga, A. Kuno, and H. R. Ueda, “Advanced CUBIC protocols for whole-brain and whole-body clearing and imaging,” Nat. Protoc. 10(11), 1709–1727 (2015).
[Crossref] [PubMed]

E. A. Susaki, K. Tainaka, D. Perrin, F. Kishino, T. Tawara, T. M. Watanabe, C. Yokoyama, H. Onoe, M. Eguchi, S. Yamaguchi, T. Abe, H. Kiyonari, Y. Shimizu, A. Miyawaki, H. Yokota, and H. R. Ueda, “Whole-Brain Imaging with Single-Cell Resolution Using Chemical Cocktails and Computational Analysis,” Cell 157(3), 726–739 (2014).
[Crossref] [PubMed]

Tasic, B.

M. D. Muzumdar, B. Tasic, K. Miyamichi, L. Li, and L. Luo, “A global double-fluorescent Cre reporter mouse,” Genesis 45(9), 593–605 (2007).
[Crossref] [PubMed]

Tawara, T.

E. A. Susaki, K. Tainaka, D. Perrin, F. Kishino, T. Tawara, T. M. Watanabe, C. Yokoyama, H. Onoe, M. Eguchi, S. Yamaguchi, T. Abe, H. Kiyonari, Y. Shimizu, A. Miyawaki, H. Yokota, and H. R. Ueda, “Whole-Brain Imaging with Single-Cell Resolution Using Chemical Cocktails and Computational Analysis,” Cell 157(3), 726–739 (2014).
[Crossref] [PubMed]

Ueda, H. R.

E. A. Susaki, K. Tainaka, D. Perrin, H. Yukinaga, A. Kuno, and H. R. Ueda, “Advanced CUBIC protocols for whole-brain and whole-body clearing and imaging,” Nat. Protoc. 10(11), 1709–1727 (2015).
[Crossref] [PubMed]

E. A. Susaki, K. Tainaka, D. Perrin, F. Kishino, T. Tawara, T. M. Watanabe, C. Yokoyama, H. Onoe, M. Eguchi, S. Yamaguchi, T. Abe, H. Kiyonari, Y. Shimizu, A. Miyawaki, H. Yokota, and H. R. Ueda, “Whole-Brain Imaging with Single-Cell Resolution Using Chemical Cocktails and Computational Analysis,” Cell 157(3), 726–739 (2014).
[Crossref] [PubMed]

Watanabe, T. M.

E. A. Susaki, K. Tainaka, D. Perrin, F. Kishino, T. Tawara, T. M. Watanabe, C. Yokoyama, H. Onoe, M. Eguchi, S. Yamaguchi, T. Abe, H. Kiyonari, Y. Shimizu, A. Miyawaki, H. Yokota, and H. R. Ueda, “Whole-Brain Imaging with Single-Cell Resolution Using Chemical Cocktails and Computational Analysis,” Cell 157(3), 726–739 (2014).
[Crossref] [PubMed]

Yamaguchi, S.

E. A. Susaki, K. Tainaka, D. Perrin, F. Kishino, T. Tawara, T. M. Watanabe, C. Yokoyama, H. Onoe, M. Eguchi, S. Yamaguchi, T. Abe, H. Kiyonari, Y. Shimizu, A. Miyawaki, H. Yokota, and H. R. Ueda, “Whole-Brain Imaging with Single-Cell Resolution Using Chemical Cocktails and Computational Analysis,” Cell 157(3), 726–739 (2014).
[Crossref] [PubMed]

Yokota, H.

E. A. Susaki, K. Tainaka, D. Perrin, F. Kishino, T. Tawara, T. M. Watanabe, C. Yokoyama, H. Onoe, M. Eguchi, S. Yamaguchi, T. Abe, H. Kiyonari, Y. Shimizu, A. Miyawaki, H. Yokota, and H. R. Ueda, “Whole-Brain Imaging with Single-Cell Resolution Using Chemical Cocktails and Computational Analysis,” Cell 157(3), 726–739 (2014).
[Crossref] [PubMed]

Yokoyama, C.

E. A. Susaki, K. Tainaka, D. Perrin, F. Kishino, T. Tawara, T. M. Watanabe, C. Yokoyama, H. Onoe, M. Eguchi, S. Yamaguchi, T. Abe, H. Kiyonari, Y. Shimizu, A. Miyawaki, H. Yokota, and H. R. Ueda, “Whole-Brain Imaging with Single-Cell Resolution Using Chemical Cocktails and Computational Analysis,” Cell 157(3), 726–739 (2014).
[Crossref] [PubMed]

Yukinaga, H.

E. A. Susaki, K. Tainaka, D. Perrin, H. Yukinaga, A. Kuno, and H. R. Ueda, “Advanced CUBIC protocols for whole-brain and whole-body clearing and imaging,” Nat. Protoc. 10(11), 1709–1727 (2015).
[Crossref] [PubMed]

Biomed. Opt. Express (1)

Biotechniques (1)

M. Sivaguru, G. Fried, B. S. Sivaguru, V. A. Sivaguru, X. Lu, K. H. Choi, M. T. Saif, B. Lin, and S. Sadayappan, “Cardiac muscle organization revealed in 3-D by imaging whole-mount mouse hearts using two-photon fluorescence and confocal microscopy,” Biotechniques 59(5), 295–308 (2015).
[Crossref] [PubMed]

Cell (2)

D. S. Richardson and J. W. Lichtman, “Clarifying Tissue Clearing,” Cell 162(2), 246–257 (2015).
[Crossref] [PubMed]

E. A. Susaki, K. Tainaka, D. Perrin, F. Kishino, T. Tawara, T. M. Watanabe, C. Yokoyama, H. Onoe, M. Eguchi, S. Yamaguchi, T. Abe, H. Kiyonari, Y. Shimizu, A. Miyawaki, H. Yokota, and H. R. Ueda, “Whole-Brain Imaging with Single-Cell Resolution Using Chemical Cocktails and Computational Analysis,” Cell 157(3), 726–739 (2014).
[Crossref] [PubMed]

Genesis (1)

M. D. Muzumdar, B. Tasic, K. Miyamichi, L. Li, and L. Luo, “A global double-fluorescent Cre reporter mouse,” Genesis 45(9), 593–605 (2007).
[Crossref] [PubMed]

J. Cardiovasc. Transl. Res. (2)

A. Pasipoularides, “Mechanotransduction Mechanisms for Intraventricular Diastolic Vortex Forces and Myocardial Deformations: Part 1,” J. Cardiovasc. Transl. Res. 8(1), 76–87 (2015).
[Crossref] [PubMed]

A. Pasipoularides, “Mechanotransduction Mechanisms for Intraventricular Diastolic Vortex Forces and Myocardial Deformations: Part 2,” J. Cardiovasc. Transl. Res. 8(5), 293–318 (2015).
[Crossref] [PubMed]

Nat. Protoc. (1)

E. A. Susaki, K. Tainaka, D. Perrin, H. Yukinaga, A. Kuno, and H. R. Ueda, “Advanced CUBIC protocols for whole-brain and whole-body clearing and imaging,” Nat. Protoc. 10(11), 1709–1727 (2015).
[Crossref] [PubMed]

Supplementary Material (4)

NameDescription
» Visualization 1: MP4 (631 KB)      Video 1. 10X non-cleared transgenic mouse heart
» Visualization 2: MP4 (1633 KB)      Video 2. 10X cleared tissue transgenic mouse heart
» Visualization 3: MP4 (591 KB)      Video 3. 20X non-cleared tissue IHC-stained mouse heart IHC-stained mouse heart
» Visualization 4: MP4 (1259 KB)      Video 4. 20X cleared tissue IHC-stained mouse heart

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Figures (2)

Fig. 1
Fig. 1

The modified CUBIC protocol significantly enhances light penetration into cardiac tissue. A, Heart tissue section (750 µm-thick) from LysMcre+/−,mT/mG transgenic mice were perfused with a standard protocol. B, C, Confocal images in the XY plane and their corresponding reconstructions in the XZ plane were obtained using a 10x (B) and a 20x (C) objective. Red, TdTomato; Blue, DAPI. The depth of the tissue in the XZ plane is indicated in microns. D, Heart tissue section (750 µm-thick) from LysMcre+/−,mT/mG transgenic mice perfused following the modified CUBIC protocol. The background text allows to appreciate the transparency of the tissue. E, F, Confocal images in the XY and XZ planes were obtained using a 10x (E) or a 20x (F) objective. G, H, Z-profile mean intensity distribution ± SD thresholded DAPI channel of the images acquired with 10x (G) and 20x magnification (H). Scale bars in all the images: 50µm. (I),The area under curve of the Z-profile (mean intensity distribution ± SD) of thresholded DAPI channel of the images acquired of the cleared (blue) and non-cleared (orange) heart samples with 10x and 20x. n = 4 heart samples per condition· *p<0.05, Mann-Whitney test.

Fig. 2
Fig. 2

The modified CUBIC protocol improves antibody penetrance into cardiac tissue sections A, B, Non-cleared sections (A) and sections cleared using the modified CUBIC protocol (B) were immunostained using an anti-CD31 antibody and confocal images of the XY plane and the corresponding XZ plane were obtained. C, 3D view of the vasculature of the CUBIC-cleared tissue. Red, anti-CD31; Blue, DAPI. Scale bars in all images: 50 µm.

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