Abstract

Three-dimensional cell biology and histology of tissue sections strongly benefit from advanced light microscopy and optimized staining procedures to gather the full three-dimensional information. In particular, the combination of optical clearing with light sheet-based fluorescence microscopy simplifies fast high-quality imaging of thick biological specimens. However, verified in toto immunostaining protocols for large multicellular spheroids or for tissue sections have not been published. We present a method for the verification of immunostaining in three-dimensional spheroids. The analysis relies on three criteria to evaluate the immunostaining quality: quality of the antibody stain specificity, signal intensity achieved by the staining procedure and the correlation of the signal intensity with that of a homogeneously dispersed fluorescent dye. We optimized and investigated variations of five immunostaining protocols for three-dimensional cell biology. Our method is an important contribution to three-dimensional cell biology and the histology of tissues since it allows to evaluate the efficiency of immunostaining protocols for large three-dimensional specimens, and to study the distribution of protein expression and cell types within spheroids and spheroid-specific morphological structures without the need of physical sectioning.

© 2017 Optical Society of America

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References

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  4. J. Friedrich, R. Ebner, and L. A. Kunz-Schughart, “Experimental anti-tumor therapy in 3-D: spheroids--old hat or new challenge?” Int. J. Radiat. Biol. 83(11-12), 849–871 (2007).
    [Crossref] [PubMed]
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    [Crossref] [PubMed]
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    [Crossref] [PubMed]
  25. J. Ries, C. Kaplan, E. Platonova, H. Eghlidi, and H. Ewers, “A simple, versatile method for GFP-based super-resolution microscopy via nanobodies,” Nat. Methods 9(6), 582–584 (2012).
    [Crossref] [PubMed]
  26. E. Murray, J. H. Cho, D. Goodwin, T. Ku, J. Swaney, S. Y. Kim, H. Choi, Y. G. Park, J. Y. Park, A. Hubbert, M. McCue, S. Vassallo, N. Bakh, M. P. Frosch, H. S. Seung, V. J. Wedeen, and K. Chung, “Simple, Scalable Proteomic Imaging for High-Dimensional Profiling of Intact Systems,” Cell 163(6), 1500–1514 (2015).
    [Crossref] [PubMed]
  27. K. Chung and K. Deisseroth, “CLARITY for mapping the nervous system,” Nat. Methods 10(6), 508–513 (2013).
    [Crossref] [PubMed]

2015 (2)

B. Mathew, A. Schmitz, S. Muñoz-Descalzo, N. Ansari, F. Pampaloni, E. H. K. Stelzer, and S. C. Fischer, “Robust and automated three-dimensional segmentation of densely packed cell nuclei in different biological specimens with Lines-of-Sight decomposition,” BMC Bioinformatics 16(1), 187 (2015).
[Crossref] [PubMed]

E. Murray, J. H. Cho, D. Goodwin, T. Ku, J. Swaney, S. Y. Kim, H. Choi, Y. G. Park, J. Y. Park, A. Hubbert, M. McCue, S. Vassallo, N. Bakh, M. P. Frosch, H. S. Seung, V. J. Wedeen, and K. Chung, “Simple, Scalable Proteomic Imaging for High-Dimensional Profiling of Intact Systems,” Cell 163(6), 1500–1514 (2015).
[Crossref] [PubMed]

2014 (1)

E. H. K. Stelzer, “Light-sheet fluorescence microscopy for quantitative biology,” Nat. Methods 12(1), 23–26 (2014).
[Crossref] [PubMed]

2013 (1)

K. Chung and K. Deisseroth, “CLARITY for mapping the nervous system,” Nat. Methods 10(6), 508–513 (2013).
[Crossref] [PubMed]

2012 (4)

U. Schnell, F. Dijk, K. A. Sjollema, and B. N. G. Giepmans, “Immunolabeling artifacts and the need for live-cell imaging,” Nat. Methods 9(2), 152–158 (2012).
[Crossref] [PubMed]

J. Ries, C. Kaplan, E. Platonova, H. Eghlidi, and H. Ewers, “A simple, versatile method for GFP-based super-resolution microscopy via nanobodies,” Nat. Methods 9(6), 582–584 (2012).
[Crossref] [PubMed]

I. Dufau, C. Frongia, F. Sicard, L. Dedieu, P. Cordelier, F. Ausseil, B. Ducommun, and A. Valette, “Multicellular tumor spheroid model to evaluate spatio-temporal dynamics effect of chemotherapeutics: application to the gemcitabine/CHK1 inhibitor combination in pancreatic cancer,” BMC Cancer 12(1), 15 (2012).
[Crossref] [PubMed]

P. J. Keller and H. U. Dodt, “Light sheet microscopy of living or cleared specimens,” Curr. Opin. Neurobiol. 22(1), 138–143 (2012).
[Crossref] [PubMed]

2010 (1)

L. B. Weiswald, J. M. Guinebretière, S. Richon, D. Bellet, B. Saubaméa, and V. Dangles-Marie, “In situ protein expression in tumour spheres: development of an immunostaining protocol for confocal microscopy,” BMC Cancer 10(1), 106 (2010).
[Crossref] [PubMed]

2008 (2)

P. J. Keller, A. D. Schmidt, J. Wittbrodt, and E. H. K. Stelzer, “Reconstruction of zebrafish early embryonic development by scanned light sheet microscopy,” Science 322(5904), 1065–1069 (2008).
[Crossref] [PubMed]

S. R. Ott, “Confocal microscopy in large insect brains: zinc-formaldehyde fixation improves synapsin immunostaining and preservation of morphology in whole-mounts,” J. Neurosci. Methods 172(2), 220–230 (2008).
[Crossref] [PubMed]

2007 (2)

H. U. Dodt, U. Leischner, A. Schierloh, N. Jährling, C. P. Mauch, K. Deininger, J. M. Deussing, M. Eder, W. Zieglgänsberger, and K. Becker, “Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain,” Nat. Methods 4(4), 331–336 (2007).
[Crossref] [PubMed]

J. Friedrich, R. Ebner, and L. A. Kunz-Schughart, “Experimental anti-tumor therapy in 3-D: spheroids--old hat or new challenge?” Int. J. Radiat. Biol. 83(11-12), 849–871 (2007).
[Crossref] [PubMed]

2005 (1)

H. R. Mellor, D. J. P. Ferguson, and R. Callaghan, “A model of quiescent tumour microregions for evaluating multicellular resistance to chemotherapeutic drugs,” Br. J. Cancer 93(3), 302–309 (2005).
[Crossref] [PubMed]

2003 (1)

J. Debnath, S. K. Muthuswamy, and J. S. Brugge, “Morphogenesis and oncogenesis of MCF-10A mammary epithelial acini grown in three-dimensional basement membrane cultures,” Methods 30(3), 256–268 (2003).
[Crossref] [PubMed]

2001 (1)

H. S. Bell, I. R. Whittle, M. Walker, H. A. Leaver, and S. B. Wharton, “The development of necrosis and apoptosis in glioma: experimental findings using spheroid culture systems,” Neuropathol. Appl. Neurobiol. 27(4), 291–304 (2001).
[Crossref] [PubMed]

1999 (1)

M. T. Santini and G. Rainaldi, “Three-dimensional spheroid model in tumor biology,” Pathobiology 67(3), 148–157 (1999).
[Crossref] [PubMed]

1998 (3)

P. Brandtzaeg, “The increasing power of immunohistochemistry and immunocytochemistry,” J. Immunol. Methods 216(1-2), 49–67 (1998).
[Crossref] [PubMed]

E. H. K. Stelzer, “Contrast, resolution, pixelation, dynamic range and signal-to-noise ratio: fundamental limits to resolution in fluorescence light microscopy,” J. Microsc. 189(1), 15–24 (1998).
[Crossref]

D. C. Altieri, F. Li, G. Ambrosini, E. Y. Chu, J. Plescia, S. Tognin, and P. C. Marchisio, “Control of apoptosis and mitotic spindle checkpoint by survivin,” Nature 396(6711), 580–584 (1998).
[Crossref] [PubMed]

1996 (1)

K. Groebe and W. Mueller-Klieser, “On the relation between size of necrosis and diameter of tumor spheroids,” Int. J. Radiat. Oncol. Biol. Phys. 34(2), 395–401 (1996).
[Crossref] [PubMed]

1994 (1)

M. G. Nichols and T. H. Foster, “Oxygen diffusion and reaction kinetics in the photodynamic therapy of multicell tumour spheroids,” Phys. Med. Biol. 39(12), 2161–2181 (1994).
[Crossref] [PubMed]

1989 (1)

J. A. Dent, A. G. Polson, and M. W. Klymkowsky, “A whole-mount immunocytochemical analysis of the expression of the intermediate filament protein vimentin in Xenopus,” Development 105(1), 61–74 (1989).
[PubMed]

1988 (1)

R. M. Sutherland, “Cell and environment interactions in tumor microregions: the multicell spheroid model,” Science 240(4849), 177–184 (1988).
[Crossref] [PubMed]

1984 (1)

W. Mueller-Klieser, “Method for the determination of oxygen consumption rates and diffusion coefficients in multicellular spheroids,” Biophys. J. 46(3), 343–348 (1984).
[Crossref] [PubMed]

1949 (1)

A. H. Coons and M. H. Kaplan, “Localization of antigen in tissue cells; improvements in a method for the detection of antigen by means of fluorescent antibody,” J. Exp. Med. 91(1), 1–13 (1949).
[Crossref] [PubMed]

Altieri, D. C.

D. C. Altieri, F. Li, G. Ambrosini, E. Y. Chu, J. Plescia, S. Tognin, and P. C. Marchisio, “Control of apoptosis and mitotic spindle checkpoint by survivin,” Nature 396(6711), 580–584 (1998).
[Crossref] [PubMed]

Ambrosini, G.

D. C. Altieri, F. Li, G. Ambrosini, E. Y. Chu, J. Plescia, S. Tognin, and P. C. Marchisio, “Control of apoptosis and mitotic spindle checkpoint by survivin,” Nature 396(6711), 580–584 (1998).
[Crossref] [PubMed]

Ansari, N.

B. Mathew, A. Schmitz, S. Muñoz-Descalzo, N. Ansari, F. Pampaloni, E. H. K. Stelzer, and S. C. Fischer, “Robust and automated three-dimensional segmentation of densely packed cell nuclei in different biological specimens with Lines-of-Sight decomposition,” BMC Bioinformatics 16(1), 187 (2015).
[Crossref] [PubMed]

Ausseil, F.

I. Dufau, C. Frongia, F. Sicard, L. Dedieu, P. Cordelier, F. Ausseil, B. Ducommun, and A. Valette, “Multicellular tumor spheroid model to evaluate spatio-temporal dynamics effect of chemotherapeutics: application to the gemcitabine/CHK1 inhibitor combination in pancreatic cancer,” BMC Cancer 12(1), 15 (2012).
[Crossref] [PubMed]

Bakh, N.

E. Murray, J. H. Cho, D. Goodwin, T. Ku, J. Swaney, S. Y. Kim, H. Choi, Y. G. Park, J. Y. Park, A. Hubbert, M. McCue, S. Vassallo, N. Bakh, M. P. Frosch, H. S. Seung, V. J. Wedeen, and K. Chung, “Simple, Scalable Proteomic Imaging for High-Dimensional Profiling of Intact Systems,” Cell 163(6), 1500–1514 (2015).
[Crossref] [PubMed]

Becker, K.

H. U. Dodt, U. Leischner, A. Schierloh, N. Jährling, C. P. Mauch, K. Deininger, J. M. Deussing, M. Eder, W. Zieglgänsberger, and K. Becker, “Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain,” Nat. Methods 4(4), 331–336 (2007).
[Crossref] [PubMed]

Bell, H. S.

H. S. Bell, I. R. Whittle, M. Walker, H. A. Leaver, and S. B. Wharton, “The development of necrosis and apoptosis in glioma: experimental findings using spheroid culture systems,” Neuropathol. Appl. Neurobiol. 27(4), 291–304 (2001).
[Crossref] [PubMed]

Bellet, D.

L. B. Weiswald, J. M. Guinebretière, S. Richon, D. Bellet, B. Saubaméa, and V. Dangles-Marie, “In situ protein expression in tumour spheres: development of an immunostaining protocol for confocal microscopy,” BMC Cancer 10(1), 106 (2010).
[Crossref] [PubMed]

Brandtzaeg, P.

P. Brandtzaeg, “The increasing power of immunohistochemistry and immunocytochemistry,” J. Immunol. Methods 216(1-2), 49–67 (1998).
[Crossref] [PubMed]

Brugge, J. S.

J. Debnath, S. K. Muthuswamy, and J. S. Brugge, “Morphogenesis and oncogenesis of MCF-10A mammary epithelial acini grown in three-dimensional basement membrane cultures,” Methods 30(3), 256–268 (2003).
[Crossref] [PubMed]

Callaghan, R.

H. R. Mellor, D. J. P. Ferguson, and R. Callaghan, “A model of quiescent tumour microregions for evaluating multicellular resistance to chemotherapeutic drugs,” Br. J. Cancer 93(3), 302–309 (2005).
[Crossref] [PubMed]

Cho, J. H.

E. Murray, J. H. Cho, D. Goodwin, T. Ku, J. Swaney, S. Y. Kim, H. Choi, Y. G. Park, J. Y. Park, A. Hubbert, M. McCue, S. Vassallo, N. Bakh, M. P. Frosch, H. S. Seung, V. J. Wedeen, and K. Chung, “Simple, Scalable Proteomic Imaging for High-Dimensional Profiling of Intact Systems,” Cell 163(6), 1500–1514 (2015).
[Crossref] [PubMed]

Choi, H.

E. Murray, J. H. Cho, D. Goodwin, T. Ku, J. Swaney, S. Y. Kim, H. Choi, Y. G. Park, J. Y. Park, A. Hubbert, M. McCue, S. Vassallo, N. Bakh, M. P. Frosch, H. S. Seung, V. J. Wedeen, and K. Chung, “Simple, Scalable Proteomic Imaging for High-Dimensional Profiling of Intact Systems,” Cell 163(6), 1500–1514 (2015).
[Crossref] [PubMed]

Chu, E. Y.

D. C. Altieri, F. Li, G. Ambrosini, E. Y. Chu, J. Plescia, S. Tognin, and P. C. Marchisio, “Control of apoptosis and mitotic spindle checkpoint by survivin,” Nature 396(6711), 580–584 (1998).
[Crossref] [PubMed]

Chung, K.

E. Murray, J. H. Cho, D. Goodwin, T. Ku, J. Swaney, S. Y. Kim, H. Choi, Y. G. Park, J. Y. Park, A. Hubbert, M. McCue, S. Vassallo, N. Bakh, M. P. Frosch, H. S. Seung, V. J. Wedeen, and K. Chung, “Simple, Scalable Proteomic Imaging for High-Dimensional Profiling of Intact Systems,” Cell 163(6), 1500–1514 (2015).
[Crossref] [PubMed]

K. Chung and K. Deisseroth, “CLARITY for mapping the nervous system,” Nat. Methods 10(6), 508–513 (2013).
[Crossref] [PubMed]

Coons, A. H.

A. H. Coons and M. H. Kaplan, “Localization of antigen in tissue cells; improvements in a method for the detection of antigen by means of fluorescent antibody,” J. Exp. Med. 91(1), 1–13 (1949).
[Crossref] [PubMed]

Cordelier, P.

I. Dufau, C. Frongia, F. Sicard, L. Dedieu, P. Cordelier, F. Ausseil, B. Ducommun, and A. Valette, “Multicellular tumor spheroid model to evaluate spatio-temporal dynamics effect of chemotherapeutics: application to the gemcitabine/CHK1 inhibitor combination in pancreatic cancer,” BMC Cancer 12(1), 15 (2012).
[Crossref] [PubMed]

Dangles-Marie, V.

L. B. Weiswald, J. M. Guinebretière, S. Richon, D. Bellet, B. Saubaméa, and V. Dangles-Marie, “In situ protein expression in tumour spheres: development of an immunostaining protocol for confocal microscopy,” BMC Cancer 10(1), 106 (2010).
[Crossref] [PubMed]

Debnath, J.

J. Debnath, S. K. Muthuswamy, and J. S. Brugge, “Morphogenesis and oncogenesis of MCF-10A mammary epithelial acini grown in three-dimensional basement membrane cultures,” Methods 30(3), 256–268 (2003).
[Crossref] [PubMed]

Dedieu, L.

I. Dufau, C. Frongia, F. Sicard, L. Dedieu, P. Cordelier, F. Ausseil, B. Ducommun, and A. Valette, “Multicellular tumor spheroid model to evaluate spatio-temporal dynamics effect of chemotherapeutics: application to the gemcitabine/CHK1 inhibitor combination in pancreatic cancer,” BMC Cancer 12(1), 15 (2012).
[Crossref] [PubMed]

Deininger, K.

H. U. Dodt, U. Leischner, A. Schierloh, N. Jährling, C. P. Mauch, K. Deininger, J. M. Deussing, M. Eder, W. Zieglgänsberger, and K. Becker, “Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain,” Nat. Methods 4(4), 331–336 (2007).
[Crossref] [PubMed]

Deisseroth, K.

K. Chung and K. Deisseroth, “CLARITY for mapping the nervous system,” Nat. Methods 10(6), 508–513 (2013).
[Crossref] [PubMed]

Dent, J. A.

J. A. Dent, A. G. Polson, and M. W. Klymkowsky, “A whole-mount immunocytochemical analysis of the expression of the intermediate filament protein vimentin in Xenopus,” Development 105(1), 61–74 (1989).
[PubMed]

Deussing, J. M.

H. U. Dodt, U. Leischner, A. Schierloh, N. Jährling, C. P. Mauch, K. Deininger, J. M. Deussing, M. Eder, W. Zieglgänsberger, and K. Becker, “Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain,” Nat. Methods 4(4), 331–336 (2007).
[Crossref] [PubMed]

Dijk, F.

U. Schnell, F. Dijk, K. A. Sjollema, and B. N. G. Giepmans, “Immunolabeling artifacts and the need for live-cell imaging,” Nat. Methods 9(2), 152–158 (2012).
[Crossref] [PubMed]

Dodt, H. U.

P. J. Keller and H. U. Dodt, “Light sheet microscopy of living or cleared specimens,” Curr. Opin. Neurobiol. 22(1), 138–143 (2012).
[Crossref] [PubMed]

H. U. Dodt, U. Leischner, A. Schierloh, N. Jährling, C. P. Mauch, K. Deininger, J. M. Deussing, M. Eder, W. Zieglgänsberger, and K. Becker, “Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain,” Nat. Methods 4(4), 331–336 (2007).
[Crossref] [PubMed]

Ducommun, B.

I. Dufau, C. Frongia, F. Sicard, L. Dedieu, P. Cordelier, F. Ausseil, B. Ducommun, and A. Valette, “Multicellular tumor spheroid model to evaluate spatio-temporal dynamics effect of chemotherapeutics: application to the gemcitabine/CHK1 inhibitor combination in pancreatic cancer,” BMC Cancer 12(1), 15 (2012).
[Crossref] [PubMed]

Dufau, I.

I. Dufau, C. Frongia, F. Sicard, L. Dedieu, P. Cordelier, F. Ausseil, B. Ducommun, and A. Valette, “Multicellular tumor spheroid model to evaluate spatio-temporal dynamics effect of chemotherapeutics: application to the gemcitabine/CHK1 inhibitor combination in pancreatic cancer,” BMC Cancer 12(1), 15 (2012).
[Crossref] [PubMed]

Ebner, R.

J. Friedrich, R. Ebner, and L. A. Kunz-Schughart, “Experimental anti-tumor therapy in 3-D: spheroids--old hat or new challenge?” Int. J. Radiat. Biol. 83(11-12), 849–871 (2007).
[Crossref] [PubMed]

Eder, M.

H. U. Dodt, U. Leischner, A. Schierloh, N. Jährling, C. P. Mauch, K. Deininger, J. M. Deussing, M. Eder, W. Zieglgänsberger, and K. Becker, “Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain,” Nat. Methods 4(4), 331–336 (2007).
[Crossref] [PubMed]

Eghlidi, H.

J. Ries, C. Kaplan, E. Platonova, H. Eghlidi, and H. Ewers, “A simple, versatile method for GFP-based super-resolution microscopy via nanobodies,” Nat. Methods 9(6), 582–584 (2012).
[Crossref] [PubMed]

Ewers, H.

J. Ries, C. Kaplan, E. Platonova, H. Eghlidi, and H. Ewers, “A simple, versatile method for GFP-based super-resolution microscopy via nanobodies,” Nat. Methods 9(6), 582–584 (2012).
[Crossref] [PubMed]

Ferguson, D. J. P.

H. R. Mellor, D. J. P. Ferguson, and R. Callaghan, “A model of quiescent tumour microregions for evaluating multicellular resistance to chemotherapeutic drugs,” Br. J. Cancer 93(3), 302–309 (2005).
[Crossref] [PubMed]

Fischer, S. C.

B. Mathew, A. Schmitz, S. Muñoz-Descalzo, N. Ansari, F. Pampaloni, E. H. K. Stelzer, and S. C. Fischer, “Robust and automated three-dimensional segmentation of densely packed cell nuclei in different biological specimens with Lines-of-Sight decomposition,” BMC Bioinformatics 16(1), 187 (2015).
[Crossref] [PubMed]

Foster, T. H.

M. G. Nichols and T. H. Foster, “Oxygen diffusion and reaction kinetics in the photodynamic therapy of multicell tumour spheroids,” Phys. Med. Biol. 39(12), 2161–2181 (1994).
[Crossref] [PubMed]

Friedrich, J.

J. Friedrich, R. Ebner, and L. A. Kunz-Schughart, “Experimental anti-tumor therapy in 3-D: spheroids--old hat or new challenge?” Int. J. Radiat. Biol. 83(11-12), 849–871 (2007).
[Crossref] [PubMed]

Frongia, C.

I. Dufau, C. Frongia, F. Sicard, L. Dedieu, P. Cordelier, F. Ausseil, B. Ducommun, and A. Valette, “Multicellular tumor spheroid model to evaluate spatio-temporal dynamics effect of chemotherapeutics: application to the gemcitabine/CHK1 inhibitor combination in pancreatic cancer,” BMC Cancer 12(1), 15 (2012).
[Crossref] [PubMed]

Frosch, M. P.

E. Murray, J. H. Cho, D. Goodwin, T. Ku, J. Swaney, S. Y. Kim, H. Choi, Y. G. Park, J. Y. Park, A. Hubbert, M. McCue, S. Vassallo, N. Bakh, M. P. Frosch, H. S. Seung, V. J. Wedeen, and K. Chung, “Simple, Scalable Proteomic Imaging for High-Dimensional Profiling of Intact Systems,” Cell 163(6), 1500–1514 (2015).
[Crossref] [PubMed]

Giepmans, B. N. G.

U. Schnell, F. Dijk, K. A. Sjollema, and B. N. G. Giepmans, “Immunolabeling artifacts and the need for live-cell imaging,” Nat. Methods 9(2), 152–158 (2012).
[Crossref] [PubMed]

Goodwin, D.

E. Murray, J. H. Cho, D. Goodwin, T. Ku, J. Swaney, S. Y. Kim, H. Choi, Y. G. Park, J. Y. Park, A. Hubbert, M. McCue, S. Vassallo, N. Bakh, M. P. Frosch, H. S. Seung, V. J. Wedeen, and K. Chung, “Simple, Scalable Proteomic Imaging for High-Dimensional Profiling of Intact Systems,” Cell 163(6), 1500–1514 (2015).
[Crossref] [PubMed]

Groebe, K.

K. Groebe and W. Mueller-Klieser, “On the relation between size of necrosis and diameter of tumor spheroids,” Int. J. Radiat. Oncol. Biol. Phys. 34(2), 395–401 (1996).
[Crossref] [PubMed]

Guinebretière, J. M.

L. B. Weiswald, J. M. Guinebretière, S. Richon, D. Bellet, B. Saubaméa, and V. Dangles-Marie, “In situ protein expression in tumour spheres: development of an immunostaining protocol for confocal microscopy,” BMC Cancer 10(1), 106 (2010).
[Crossref] [PubMed]

Hubbert, A.

E. Murray, J. H. Cho, D. Goodwin, T. Ku, J. Swaney, S. Y. Kim, H. Choi, Y. G. Park, J. Y. Park, A. Hubbert, M. McCue, S. Vassallo, N. Bakh, M. P. Frosch, H. S. Seung, V. J. Wedeen, and K. Chung, “Simple, Scalable Proteomic Imaging for High-Dimensional Profiling of Intact Systems,” Cell 163(6), 1500–1514 (2015).
[Crossref] [PubMed]

Jährling, N.

H. U. Dodt, U. Leischner, A. Schierloh, N. Jährling, C. P. Mauch, K. Deininger, J. M. Deussing, M. Eder, W. Zieglgänsberger, and K. Becker, “Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain,” Nat. Methods 4(4), 331–336 (2007).
[Crossref] [PubMed]

Kaplan, C.

J. Ries, C. Kaplan, E. Platonova, H. Eghlidi, and H. Ewers, “A simple, versatile method for GFP-based super-resolution microscopy via nanobodies,” Nat. Methods 9(6), 582–584 (2012).
[Crossref] [PubMed]

Kaplan, M. H.

A. H. Coons and M. H. Kaplan, “Localization of antigen in tissue cells; improvements in a method for the detection of antigen by means of fluorescent antibody,” J. Exp. Med. 91(1), 1–13 (1949).
[Crossref] [PubMed]

Keller, P. J.

P. J. Keller and H. U. Dodt, “Light sheet microscopy of living or cleared specimens,” Curr. Opin. Neurobiol. 22(1), 138–143 (2012).
[Crossref] [PubMed]

P. J. Keller, A. D. Schmidt, J. Wittbrodt, and E. H. K. Stelzer, “Reconstruction of zebrafish early embryonic development by scanned light sheet microscopy,” Science 322(5904), 1065–1069 (2008).
[Crossref] [PubMed]

Kim, S. Y.

E. Murray, J. H. Cho, D. Goodwin, T. Ku, J. Swaney, S. Y. Kim, H. Choi, Y. G. Park, J. Y. Park, A. Hubbert, M. McCue, S. Vassallo, N. Bakh, M. P. Frosch, H. S. Seung, V. J. Wedeen, and K. Chung, “Simple, Scalable Proteomic Imaging for High-Dimensional Profiling of Intact Systems,” Cell 163(6), 1500–1514 (2015).
[Crossref] [PubMed]

Klymkowsky, M. W.

J. A. Dent, A. G. Polson, and M. W. Klymkowsky, “A whole-mount immunocytochemical analysis of the expression of the intermediate filament protein vimentin in Xenopus,” Development 105(1), 61–74 (1989).
[PubMed]

Ku, T.

E. Murray, J. H. Cho, D. Goodwin, T. Ku, J. Swaney, S. Y. Kim, H. Choi, Y. G. Park, J. Y. Park, A. Hubbert, M. McCue, S. Vassallo, N. Bakh, M. P. Frosch, H. S. Seung, V. J. Wedeen, and K. Chung, “Simple, Scalable Proteomic Imaging for High-Dimensional Profiling of Intact Systems,” Cell 163(6), 1500–1514 (2015).
[Crossref] [PubMed]

Kunz-Schughart, L. A.

J. Friedrich, R. Ebner, and L. A. Kunz-Schughart, “Experimental anti-tumor therapy in 3-D: spheroids--old hat or new challenge?” Int. J. Radiat. Biol. 83(11-12), 849–871 (2007).
[Crossref] [PubMed]

Leaver, H. A.

H. S. Bell, I. R. Whittle, M. Walker, H. A. Leaver, and S. B. Wharton, “The development of necrosis and apoptosis in glioma: experimental findings using spheroid culture systems,” Neuropathol. Appl. Neurobiol. 27(4), 291–304 (2001).
[Crossref] [PubMed]

Leischner, U.

H. U. Dodt, U. Leischner, A. Schierloh, N. Jährling, C. P. Mauch, K. Deininger, J. M. Deussing, M. Eder, W. Zieglgänsberger, and K. Becker, “Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain,” Nat. Methods 4(4), 331–336 (2007).
[Crossref] [PubMed]

Li, F.

D. C. Altieri, F. Li, G. Ambrosini, E. Y. Chu, J. Plescia, S. Tognin, and P. C. Marchisio, “Control of apoptosis and mitotic spindle checkpoint by survivin,” Nature 396(6711), 580–584 (1998).
[Crossref] [PubMed]

Marchisio, P. C.

D. C. Altieri, F. Li, G. Ambrosini, E. Y. Chu, J. Plescia, S. Tognin, and P. C. Marchisio, “Control of apoptosis and mitotic spindle checkpoint by survivin,” Nature 396(6711), 580–584 (1998).
[Crossref] [PubMed]

Mathew, B.

B. Mathew, A. Schmitz, S. Muñoz-Descalzo, N. Ansari, F. Pampaloni, E. H. K. Stelzer, and S. C. Fischer, “Robust and automated three-dimensional segmentation of densely packed cell nuclei in different biological specimens with Lines-of-Sight decomposition,” BMC Bioinformatics 16(1), 187 (2015).
[Crossref] [PubMed]

Mauch, C. P.

H. U. Dodt, U. Leischner, A. Schierloh, N. Jährling, C. P. Mauch, K. Deininger, J. M. Deussing, M. Eder, W. Zieglgänsberger, and K. Becker, “Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain,” Nat. Methods 4(4), 331–336 (2007).
[Crossref] [PubMed]

McCue, M.

E. Murray, J. H. Cho, D. Goodwin, T. Ku, J. Swaney, S. Y. Kim, H. Choi, Y. G. Park, J. Y. Park, A. Hubbert, M. McCue, S. Vassallo, N. Bakh, M. P. Frosch, H. S. Seung, V. J. Wedeen, and K. Chung, “Simple, Scalable Proteomic Imaging for High-Dimensional Profiling of Intact Systems,” Cell 163(6), 1500–1514 (2015).
[Crossref] [PubMed]

Mellor, H. R.

H. R. Mellor, D. J. P. Ferguson, and R. Callaghan, “A model of quiescent tumour microregions for evaluating multicellular resistance to chemotherapeutic drugs,” Br. J. Cancer 93(3), 302–309 (2005).
[Crossref] [PubMed]

Mueller-Klieser, W.

K. Groebe and W. Mueller-Klieser, “On the relation between size of necrosis and diameter of tumor spheroids,” Int. J. Radiat. Oncol. Biol. Phys. 34(2), 395–401 (1996).
[Crossref] [PubMed]

W. Mueller-Klieser, “Method for the determination of oxygen consumption rates and diffusion coefficients in multicellular spheroids,” Biophys. J. 46(3), 343–348 (1984).
[Crossref] [PubMed]

Muñoz-Descalzo, S.

B. Mathew, A. Schmitz, S. Muñoz-Descalzo, N. Ansari, F. Pampaloni, E. H. K. Stelzer, and S. C. Fischer, “Robust and automated three-dimensional segmentation of densely packed cell nuclei in different biological specimens with Lines-of-Sight decomposition,” BMC Bioinformatics 16(1), 187 (2015).
[Crossref] [PubMed]

Murray, E.

E. Murray, J. H. Cho, D. Goodwin, T. Ku, J. Swaney, S. Y. Kim, H. Choi, Y. G. Park, J. Y. Park, A. Hubbert, M. McCue, S. Vassallo, N. Bakh, M. P. Frosch, H. S. Seung, V. J. Wedeen, and K. Chung, “Simple, Scalable Proteomic Imaging for High-Dimensional Profiling of Intact Systems,” Cell 163(6), 1500–1514 (2015).
[Crossref] [PubMed]

Muthuswamy, S. K.

J. Debnath, S. K. Muthuswamy, and J. S. Brugge, “Morphogenesis and oncogenesis of MCF-10A mammary epithelial acini grown in three-dimensional basement membrane cultures,” Methods 30(3), 256–268 (2003).
[Crossref] [PubMed]

Nichols, M. G.

M. G. Nichols and T. H. Foster, “Oxygen diffusion and reaction kinetics in the photodynamic therapy of multicell tumour spheroids,” Phys. Med. Biol. 39(12), 2161–2181 (1994).
[Crossref] [PubMed]

Ott, S. R.

S. R. Ott, “Confocal microscopy in large insect brains: zinc-formaldehyde fixation improves synapsin immunostaining and preservation of morphology in whole-mounts,” J. Neurosci. Methods 172(2), 220–230 (2008).
[Crossref] [PubMed]

Pampaloni, F.

B. Mathew, A. Schmitz, S. Muñoz-Descalzo, N. Ansari, F. Pampaloni, E. H. K. Stelzer, and S. C. Fischer, “Robust and automated three-dimensional segmentation of densely packed cell nuclei in different biological specimens with Lines-of-Sight decomposition,” BMC Bioinformatics 16(1), 187 (2015).
[Crossref] [PubMed]

Park, J. Y.

E. Murray, J. H. Cho, D. Goodwin, T. Ku, J. Swaney, S. Y. Kim, H. Choi, Y. G. Park, J. Y. Park, A. Hubbert, M. McCue, S. Vassallo, N. Bakh, M. P. Frosch, H. S. Seung, V. J. Wedeen, and K. Chung, “Simple, Scalable Proteomic Imaging for High-Dimensional Profiling of Intact Systems,” Cell 163(6), 1500–1514 (2015).
[Crossref] [PubMed]

Park, Y. G.

E. Murray, J. H. Cho, D. Goodwin, T. Ku, J. Swaney, S. Y. Kim, H. Choi, Y. G. Park, J. Y. Park, A. Hubbert, M. McCue, S. Vassallo, N. Bakh, M. P. Frosch, H. S. Seung, V. J. Wedeen, and K. Chung, “Simple, Scalable Proteomic Imaging for High-Dimensional Profiling of Intact Systems,” Cell 163(6), 1500–1514 (2015).
[Crossref] [PubMed]

Platonova, E.

J. Ries, C. Kaplan, E. Platonova, H. Eghlidi, and H. Ewers, “A simple, versatile method for GFP-based super-resolution microscopy via nanobodies,” Nat. Methods 9(6), 582–584 (2012).
[Crossref] [PubMed]

Plescia, J.

D. C. Altieri, F. Li, G. Ambrosini, E. Y. Chu, J. Plescia, S. Tognin, and P. C. Marchisio, “Control of apoptosis and mitotic spindle checkpoint by survivin,” Nature 396(6711), 580–584 (1998).
[Crossref] [PubMed]

Polson, A. G.

J. A. Dent, A. G. Polson, and M. W. Klymkowsky, “A whole-mount immunocytochemical analysis of the expression of the intermediate filament protein vimentin in Xenopus,” Development 105(1), 61–74 (1989).
[PubMed]

Rainaldi, G.

M. T. Santini and G. Rainaldi, “Three-dimensional spheroid model in tumor biology,” Pathobiology 67(3), 148–157 (1999).
[Crossref] [PubMed]

Richon, S.

L. B. Weiswald, J. M. Guinebretière, S. Richon, D. Bellet, B. Saubaméa, and V. Dangles-Marie, “In situ protein expression in tumour spheres: development of an immunostaining protocol for confocal microscopy,” BMC Cancer 10(1), 106 (2010).
[Crossref] [PubMed]

Ries, J.

J. Ries, C. Kaplan, E. Platonova, H. Eghlidi, and H. Ewers, “A simple, versatile method for GFP-based super-resolution microscopy via nanobodies,” Nat. Methods 9(6), 582–584 (2012).
[Crossref] [PubMed]

Santini, M. T.

M. T. Santini and G. Rainaldi, “Three-dimensional spheroid model in tumor biology,” Pathobiology 67(3), 148–157 (1999).
[Crossref] [PubMed]

Saubaméa, B.

L. B. Weiswald, J. M. Guinebretière, S. Richon, D. Bellet, B. Saubaméa, and V. Dangles-Marie, “In situ protein expression in tumour spheres: development of an immunostaining protocol for confocal microscopy,” BMC Cancer 10(1), 106 (2010).
[Crossref] [PubMed]

Schierloh, A.

H. U. Dodt, U. Leischner, A. Schierloh, N. Jährling, C. P. Mauch, K. Deininger, J. M. Deussing, M. Eder, W. Zieglgänsberger, and K. Becker, “Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain,” Nat. Methods 4(4), 331–336 (2007).
[Crossref] [PubMed]

Schmidt, A. D.

P. J. Keller, A. D. Schmidt, J. Wittbrodt, and E. H. K. Stelzer, “Reconstruction of zebrafish early embryonic development by scanned light sheet microscopy,” Science 322(5904), 1065–1069 (2008).
[Crossref] [PubMed]

Schmitz, A.

B. Mathew, A. Schmitz, S. Muñoz-Descalzo, N. Ansari, F. Pampaloni, E. H. K. Stelzer, and S. C. Fischer, “Robust and automated three-dimensional segmentation of densely packed cell nuclei in different biological specimens with Lines-of-Sight decomposition,” BMC Bioinformatics 16(1), 187 (2015).
[Crossref] [PubMed]

Schnell, U.

U. Schnell, F. Dijk, K. A. Sjollema, and B. N. G. Giepmans, “Immunolabeling artifacts and the need for live-cell imaging,” Nat. Methods 9(2), 152–158 (2012).
[Crossref] [PubMed]

Seung, H. S.

E. Murray, J. H. Cho, D. Goodwin, T. Ku, J. Swaney, S. Y. Kim, H. Choi, Y. G. Park, J. Y. Park, A. Hubbert, M. McCue, S. Vassallo, N. Bakh, M. P. Frosch, H. S. Seung, V. J. Wedeen, and K. Chung, “Simple, Scalable Proteomic Imaging for High-Dimensional Profiling of Intact Systems,” Cell 163(6), 1500–1514 (2015).
[Crossref] [PubMed]

Sicard, F.

I. Dufau, C. Frongia, F. Sicard, L. Dedieu, P. Cordelier, F. Ausseil, B. Ducommun, and A. Valette, “Multicellular tumor spheroid model to evaluate spatio-temporal dynamics effect of chemotherapeutics: application to the gemcitabine/CHK1 inhibitor combination in pancreatic cancer,” BMC Cancer 12(1), 15 (2012).
[Crossref] [PubMed]

Sjollema, K. A.

U. Schnell, F. Dijk, K. A. Sjollema, and B. N. G. Giepmans, “Immunolabeling artifacts and the need for live-cell imaging,” Nat. Methods 9(2), 152–158 (2012).
[Crossref] [PubMed]

Stelzer, E. H. K.

B. Mathew, A. Schmitz, S. Muñoz-Descalzo, N. Ansari, F. Pampaloni, E. H. K. Stelzer, and S. C. Fischer, “Robust and automated three-dimensional segmentation of densely packed cell nuclei in different biological specimens with Lines-of-Sight decomposition,” BMC Bioinformatics 16(1), 187 (2015).
[Crossref] [PubMed]

E. H. K. Stelzer, “Light-sheet fluorescence microscopy for quantitative biology,” Nat. Methods 12(1), 23–26 (2014).
[Crossref] [PubMed]

P. J. Keller, A. D. Schmidt, J. Wittbrodt, and E. H. K. Stelzer, “Reconstruction of zebrafish early embryonic development by scanned light sheet microscopy,” Science 322(5904), 1065–1069 (2008).
[Crossref] [PubMed]

E. H. K. Stelzer, “Contrast, resolution, pixelation, dynamic range and signal-to-noise ratio: fundamental limits to resolution in fluorescence light microscopy,” J. Microsc. 189(1), 15–24 (1998).
[Crossref]

Sutherland, R. M.

R. M. Sutherland, “Cell and environment interactions in tumor microregions: the multicell spheroid model,” Science 240(4849), 177–184 (1988).
[Crossref] [PubMed]

Swaney, J.

E. Murray, J. H. Cho, D. Goodwin, T. Ku, J. Swaney, S. Y. Kim, H. Choi, Y. G. Park, J. Y. Park, A. Hubbert, M. McCue, S. Vassallo, N. Bakh, M. P. Frosch, H. S. Seung, V. J. Wedeen, and K. Chung, “Simple, Scalable Proteomic Imaging for High-Dimensional Profiling of Intact Systems,” Cell 163(6), 1500–1514 (2015).
[Crossref] [PubMed]

Tognin, S.

D. C. Altieri, F. Li, G. Ambrosini, E. Y. Chu, J. Plescia, S. Tognin, and P. C. Marchisio, “Control of apoptosis and mitotic spindle checkpoint by survivin,” Nature 396(6711), 580–584 (1998).
[Crossref] [PubMed]

Valette, A.

I. Dufau, C. Frongia, F. Sicard, L. Dedieu, P. Cordelier, F. Ausseil, B. Ducommun, and A. Valette, “Multicellular tumor spheroid model to evaluate spatio-temporal dynamics effect of chemotherapeutics: application to the gemcitabine/CHK1 inhibitor combination in pancreatic cancer,” BMC Cancer 12(1), 15 (2012).
[Crossref] [PubMed]

Vassallo, S.

E. Murray, J. H. Cho, D. Goodwin, T. Ku, J. Swaney, S. Y. Kim, H. Choi, Y. G. Park, J. Y. Park, A. Hubbert, M. McCue, S. Vassallo, N. Bakh, M. P. Frosch, H. S. Seung, V. J. Wedeen, and K. Chung, “Simple, Scalable Proteomic Imaging for High-Dimensional Profiling of Intact Systems,” Cell 163(6), 1500–1514 (2015).
[Crossref] [PubMed]

Walker, M.

H. S. Bell, I. R. Whittle, M. Walker, H. A. Leaver, and S. B. Wharton, “The development of necrosis and apoptosis in glioma: experimental findings using spheroid culture systems,” Neuropathol. Appl. Neurobiol. 27(4), 291–304 (2001).
[Crossref] [PubMed]

Wedeen, V. J.

E. Murray, J. H. Cho, D. Goodwin, T. Ku, J. Swaney, S. Y. Kim, H. Choi, Y. G. Park, J. Y. Park, A. Hubbert, M. McCue, S. Vassallo, N. Bakh, M. P. Frosch, H. S. Seung, V. J. Wedeen, and K. Chung, “Simple, Scalable Proteomic Imaging for High-Dimensional Profiling of Intact Systems,” Cell 163(6), 1500–1514 (2015).
[Crossref] [PubMed]

Weiswald, L. B.

L. B. Weiswald, J. M. Guinebretière, S. Richon, D. Bellet, B. Saubaméa, and V. Dangles-Marie, “In situ protein expression in tumour spheres: development of an immunostaining protocol for confocal microscopy,” BMC Cancer 10(1), 106 (2010).
[Crossref] [PubMed]

Wharton, S. B.

H. S. Bell, I. R. Whittle, M. Walker, H. A. Leaver, and S. B. Wharton, “The development of necrosis and apoptosis in glioma: experimental findings using spheroid culture systems,” Neuropathol. Appl. Neurobiol. 27(4), 291–304 (2001).
[Crossref] [PubMed]

Whittle, I. R.

H. S. Bell, I. R. Whittle, M. Walker, H. A. Leaver, and S. B. Wharton, “The development of necrosis and apoptosis in glioma: experimental findings using spheroid culture systems,” Neuropathol. Appl. Neurobiol. 27(4), 291–304 (2001).
[Crossref] [PubMed]

Wittbrodt, J.

P. J. Keller, A. D. Schmidt, J. Wittbrodt, and E. H. K. Stelzer, “Reconstruction of zebrafish early embryonic development by scanned light sheet microscopy,” Science 322(5904), 1065–1069 (2008).
[Crossref] [PubMed]

Zieglgänsberger, W.

H. U. Dodt, U. Leischner, A. Schierloh, N. Jährling, C. P. Mauch, K. Deininger, J. M. Deussing, M. Eder, W. Zieglgänsberger, and K. Becker, “Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain,” Nat. Methods 4(4), 331–336 (2007).
[Crossref] [PubMed]

Biophys. J. (1)

W. Mueller-Klieser, “Method for the determination of oxygen consumption rates and diffusion coefficients in multicellular spheroids,” Biophys. J. 46(3), 343–348 (1984).
[Crossref] [PubMed]

BMC Bioinformatics (1)

B. Mathew, A. Schmitz, S. Muñoz-Descalzo, N. Ansari, F. Pampaloni, E. H. K. Stelzer, and S. C. Fischer, “Robust and automated three-dimensional segmentation of densely packed cell nuclei in different biological specimens with Lines-of-Sight decomposition,” BMC Bioinformatics 16(1), 187 (2015).
[Crossref] [PubMed]

BMC Cancer (2)

I. Dufau, C. Frongia, F. Sicard, L. Dedieu, P. Cordelier, F. Ausseil, B. Ducommun, and A. Valette, “Multicellular tumor spheroid model to evaluate spatio-temporal dynamics effect of chemotherapeutics: application to the gemcitabine/CHK1 inhibitor combination in pancreatic cancer,” BMC Cancer 12(1), 15 (2012).
[Crossref] [PubMed]

L. B. Weiswald, J. M. Guinebretière, S. Richon, D. Bellet, B. Saubaméa, and V. Dangles-Marie, “In situ protein expression in tumour spheres: development of an immunostaining protocol for confocal microscopy,” BMC Cancer 10(1), 106 (2010).
[Crossref] [PubMed]

Br. J. Cancer (1)

H. R. Mellor, D. J. P. Ferguson, and R. Callaghan, “A model of quiescent tumour microregions for evaluating multicellular resistance to chemotherapeutic drugs,” Br. J. Cancer 93(3), 302–309 (2005).
[Crossref] [PubMed]

Cell (1)

E. Murray, J. H. Cho, D. Goodwin, T. Ku, J. Swaney, S. Y. Kim, H. Choi, Y. G. Park, J. Y. Park, A. Hubbert, M. McCue, S. Vassallo, N. Bakh, M. P. Frosch, H. S. Seung, V. J. Wedeen, and K. Chung, “Simple, Scalable Proteomic Imaging for High-Dimensional Profiling of Intact Systems,” Cell 163(6), 1500–1514 (2015).
[Crossref] [PubMed]

Curr. Opin. Neurobiol. (1)

P. J. Keller and H. U. Dodt, “Light sheet microscopy of living or cleared specimens,” Curr. Opin. Neurobiol. 22(1), 138–143 (2012).
[Crossref] [PubMed]

Development (1)

J. A. Dent, A. G. Polson, and M. W. Klymkowsky, “A whole-mount immunocytochemical analysis of the expression of the intermediate filament protein vimentin in Xenopus,” Development 105(1), 61–74 (1989).
[PubMed]

Int. J. Radiat. Biol. (1)

J. Friedrich, R. Ebner, and L. A. Kunz-Schughart, “Experimental anti-tumor therapy in 3-D: spheroids--old hat or new challenge?” Int. J. Radiat. Biol. 83(11-12), 849–871 (2007).
[Crossref] [PubMed]

Int. J. Radiat. Oncol. Biol. Phys. (1)

K. Groebe and W. Mueller-Klieser, “On the relation between size of necrosis and diameter of tumor spheroids,” Int. J. Radiat. Oncol. Biol. Phys. 34(2), 395–401 (1996).
[Crossref] [PubMed]

J. Exp. Med. (1)

A. H. Coons and M. H. Kaplan, “Localization of antigen in tissue cells; improvements in a method for the detection of antigen by means of fluorescent antibody,” J. Exp. Med. 91(1), 1–13 (1949).
[Crossref] [PubMed]

J. Immunol. Methods (1)

P. Brandtzaeg, “The increasing power of immunohistochemistry and immunocytochemistry,” J. Immunol. Methods 216(1-2), 49–67 (1998).
[Crossref] [PubMed]

J. Microsc. (1)

E. H. K. Stelzer, “Contrast, resolution, pixelation, dynamic range and signal-to-noise ratio: fundamental limits to resolution in fluorescence light microscopy,” J. Microsc. 189(1), 15–24 (1998).
[Crossref]

J. Neurosci. Methods (1)

S. R. Ott, “Confocal microscopy in large insect brains: zinc-formaldehyde fixation improves synapsin immunostaining and preservation of morphology in whole-mounts,” J. Neurosci. Methods 172(2), 220–230 (2008).
[Crossref] [PubMed]

Methods (1)

J. Debnath, S. K. Muthuswamy, and J. S. Brugge, “Morphogenesis and oncogenesis of MCF-10A mammary epithelial acini grown in three-dimensional basement membrane cultures,” Methods 30(3), 256–268 (2003).
[Crossref] [PubMed]

Nat. Methods (5)

U. Schnell, F. Dijk, K. A. Sjollema, and B. N. G. Giepmans, “Immunolabeling artifacts and the need for live-cell imaging,” Nat. Methods 9(2), 152–158 (2012).
[Crossref] [PubMed]

J. Ries, C. Kaplan, E. Platonova, H. Eghlidi, and H. Ewers, “A simple, versatile method for GFP-based super-resolution microscopy via nanobodies,” Nat. Methods 9(6), 582–584 (2012).
[Crossref] [PubMed]

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Figures (7)

Fig. 1
Fig. 1

(A) Schematic illustration of the immunostaining protocols. The workflow shows the different steps of the immunofluorescence staining starting with the collection of the spheroids after six days of formation. The main steps are fixation, permeabilization, blocking, antibody incubation and optical clearing. (B) Single planes at different depths in a typical spheroid data set are shown. The right image shows a 90° rotation around the y-axis of the data set that depicts the location of the single planes. Microscope: mDSLM; illumination objective: Epiplan-Neofluar 2.5x/NA 0.06; detection objective: N-Achroplan 10x/NA 0.3; α-tubulin: 561 nm, 0.09 mW, bandpass filter 607/70; β-catenin: 488 nm, 0.134 mW, bandpass filter 525/50; DAPI: 405 nm, 0.01 mW, bandpass filter 447/55; scale bar: 100 µm. h: hours, min: minutes, RT: room temperature.

Fig. 2
Fig. 2

Immunostainings against α-tubulin and β-catenin in histological sections of U343 spheroids show a homogeneous signal throughout the whole spheroid. (A) U343 spheroids were formed for six days from 10,000 seeded cells. (B) Paraffin-embedded spheroids were cut into 4 µm thick sections and stained against α-tubulin and β-catenin. A section from the middle part of a spheroid shows a homogeneous distribution of the signal for α-tubulin as well as β-catenin. Cell nuclei were counterstained with DAPI. β-catenin is located at the plasma membranes of cells (arrow). The α-tubulin antibody specifically labels the microtubules in U343 spheroids, which is best seen in the mitotic spindle during cell division (dashed circle). Microscope: Zeiss LSM780; objective lens (upper panel): Plan-Apochromat 20x/numerical aperture (NA) 0.8; objective lens (lower panel): Plan-Neofluar 40x/NA 1.3.

Fig. 3
Fig. 3

Qualitative evaluation of antibody stain specificity in U343 spheroids at the cellular level. The visibility of the spindle apparatus during mitosis indicates a functioning α-tubulin stain. Since β-catenin attaches to the adherens junctions, the label was characterized by the visibility of the cell circumference. A specific antibody stain is marked with a green check while an unspecific stain is marked with a red cross. Microscope: mDSLM; illumination objective: Epiplan-Neofluar 2.5x/NA 0.06; detection objective: N-Achroplan 20x/NA 0.5; α-tubulin: 561 nm, 0.09 mW, bandpass filter 607/70; β-catenin: 488 nm, 0.15 mW, bandpass filter 525/50; DAPI: 405 nm, 0.01 mW, bandpass filter 447/55; scale bar: 10 µm. Ab: antibody; o.n.: overnight; h: hours.

Fig. 4
Fig. 4

The optically cleared spheroid is mounted onto the sample holder (the edge of the sample holder is indicated by the white dashed line). Following image acquisition, an average projection of the image stack is computed. At the center of the spheroid, the intensity profile along a line orthogonal to the direction of the light sheet (orange line) is measured in each channel (i.e.: α-tubulin, β-catenin and DAPI). The DAPI signal is regarded as the ideal dispersion of the fluorophores in the spheroid. The intensity profiles are smoothed to reduce morphological variations. Finally, the smoothed grey values of the antibodies are plotted against the smoothed DAPI values and a linear regression is calculated. The red circles show the location of the highlighted values in the different representation modes. The output of this analysis is the Pearson’s R value. One and zero indicates that the antibody profile matches, respectively does not match, the DAPI profile.

Fig. 5
Fig. 5

Overview of the signal intensities and the degree of stain homogeneity in spheroids obtained from different immunostaining conditions. (A) Smoothed average signal intensity plots. α-tubulin and β-catenin intensity values of all samples per condition are plotted against the pixel distance along a line through the center of the spheroid. Incubation of the secondary antibody for four hours always results in lower signal intensities compared to samples incubated with the secondary antibody overnight. (B) The ratio between the maximum intensity in an average profile and the background intensity serves as a measure of the intensity yield. Ratios with values above 20 and low variance are desirable whereas values below 10 are considered insufficient. The box-plot shows the median line and the 25 and 75 percentiles. The whiskers represent the min and max values. N [PFA-Triton-4°C] = 5, N [PFA-MetOH o.n.] = 7, N [rest] = 6. Ab: antibody, Ac: acetone, dehyd.: dehydration, EtOH: ethanol, h: hours, MetOH: methanol, o.n.: overnight.

Fig. 6
Fig. 6

(A) Single planes of immunostained U343 spheroids show the dispersion of the antibody as well as the quality of the stain. Slides were taken from the middle region of the spheroids. The z-depth is indicated for each condition. White squares indicate the magnified areas (8x) in each spheroid. Microscope: mDSLM; illumination objective: Epiplan-Neofluar 2.5x/NA 0.06; detection objective: N-Achroplan 10x/NA 0.3; α-tubulin: 561 nm, 0.09 mW, bandpass filter 607/70; β-catenin: 488 nm, 0.134 mW, bandpass filter 525/50; DAPI: 405 nm, 0.01 mW, bandpass filter 447/55; scale bar: 50 µm. (B) The influence of the fixation/permeabilization and the incubation time of the secondary antibodies is expressed as Pearson’s R. It describes the degree of similarity to the DAPI intensity profile, which represents a homogeneous dispersion of a fluorophore within a spheroid. The box-plot shows the median line and the 25 and 75 percentiles. The whiskers represent the min and max values. Ab: antibody; Ac: acetone, dehyd.: dehydration, EtOH: ethanol, h: hours, MetOH: methanol, o.n.: overnight; h: hours.

Fig. 7
Fig. 7

(A) Simplified illustration of the suggested immunofluorescence protocol for large spheroids with time specifications for every step. The overall duration from sample fixation to image acquisition consumes approximately 1.5 days. This time-saving protocol suggests sample fixation with PFA followed by detergent-based permeabilization and block of unspecific binding sites. Antibody incubation temperature at 37°C allows homogeneous diffusion of antibodies. All other steps are performed at room temperature. Following staining, large spheroids are rendered transparent by optical clearing with Murray’s clear. Cleared samples are rapidly investigated with LSFM. (B) Spheroids from the murine mammary tumor cell line 4T1 were immunolabeled according the protocol in (A). Antibodies against β-catenin and GM130, a Golgi marker, were used. A section through the central part of the cleared spheroid shows the staining success. Spheroids were formed for ten days and had an initial diameter of more than 350 µm. Microscope: mDSLM; illumination objective: Epiplan-Neofluar 2.5x/NA 0.06; detection objective: N-Achroplan 20x/NA 0.5; GM130: 561 nm, 0.09 mW, bandpass filter 607/70; β-catenin: 488 nm, 0.15 mW, bandpass filter 525/50; DAPI: 405 nm, 0.01 mW, bandpass filter 447/55; scale bar: 50 µm. RT: room temperature.

Tables (2)

Tables Icon

Table 1 Immunofluorescence protocols applied in this study.

Tables Icon

Table 2 Summary of the immunofluorescence quality analysis. A qualitative as well as the two quantitative characteristics are listed (1) to describe the specificity of the stain, (2) to estimate the signal intensity obtained from the staining protocol described by the SNB ratio and (3) to rate the homogeneity of the antibody stain described by the R-value of the similarity of the intensity profiles to the DAPI stain. Stain specificity: Green indicates that the stain for microtubules shows the spindle apparatus during mitosis and that β-catenin is located at the cell periphery. Signal intensity: Green indicates a ratio above 60, orange indicates ratios between 30 and 60 and white is used for a ratio below 30. Signal homogeneity: Green indicates above 0.75, orange indicates between 0.55 and 0.74 and, white is used for values below 0.54.

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