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Three-Dimensional Reconstruction of Murine Peyer's Patches from Immunostained Cryosections

Published online by Cambridge University Press:  04 November 2013

Sarita Ahlawat ,
Magdia De Jesus ,
Kedar Khare ,
Richard A. Cole  and
Nicholas J. Mantis
Sarita Ahlawat
Affiliation:
New York State Department of Health, Wadsworth Center, Division of Infectious Diseases, Albany, NY 12208, USA
Magdia De Jesus
Affiliation:
New York State Department of Health, Wadsworth Center, Division of Infectious Diseases, Albany, NY 12208, USA
Kedar Khare
Affiliation:
Department of Physics, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110 016, India
Richard A. Cole
Affiliation:
Advanced Light Microscopy Core, New York State Department of Health, Wadsworth Center, Albany, NY 12201, USA
Nicholas J. Mantis*
Affiliation:
New York State Department of Health, Wadsworth Center, Division of Infectious Diseases, Albany, NY 12208, USA Department of Biomedical Sciences, University at Albany, Albany, NY 12208, USA
*
*Corresponding author. E-mail: nmantis@wadsworth.org
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Abstract

Peyer's patches, macroscopic aggregates of lymphoid follicles present throughout the small intestines of humans and other mammals, are considered the gateway through which luminal dietary antigens and microbes are sampled by the mucosal immune system. The cellular make-up of Peyer's patch lymphoid follicles is not only complex, but highly dynamic, as there are at least four major cell types that are known to migrate in response to antigenic stimulation. In an effort to capture the complexity and dynamic nature of this specialized tissue, here we report the three-dimensional (3D) reconstruction of immunofluorescent-labeled mouse Peyer's patch cryosections. The technology that enabled the stacking and linear blending of serial cryosections was a novel macro for Fiji, the open source image-processing package based on ImageJ. By simultaneously labeling cryosections for surface markers CD45R, CD3, and CD11c, we provide a 3D image as well as quantitative measures of B-cell, T-cell, and dendritic cell populations at steady state and following exposure to the mucosal adjuvant cholera toxin.

Keywords

lymphoid tissueintestineimmunostainingFijilinear blendingthree-dimensional reconstruction
Type
Biological Applications
Information
Microscopy and Microanalysis , Volume 20 , Issue 1 , February 2014 , pp. 198 - 205
Copyright
Copyright © Microscopy Society of America 2014 

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Footnotes

†

These authors contributed equally to this work.

References

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Ahlawat et al. Supplementary Material

3D reconstruction of mouse PP in the presence or absence of CT. Shown are full rotations of 3D structures of mouse PP obtained from a (A) control or (B) CT-treated mouse, as described in the legend to Figure 3. B cells are labeled in blue, T-cells in green and DCs in magenta.

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Ahlawat et al. Supplementary Material

3D reconstruction from optical Z sections of PP GCs following challenge with CT. The three images (left to right) each represent 3D reconstructions of approximately 40 optical Z sections corresponding to the proximal, middle and distal segments of a single Peyer’s patch take from a mouse treated with CT, as described in the text. B cells are labeled in magenta and CD3+ T-cells are labeled in blue. These data reveal the migration of B cells from the GC toward the FAE.

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Ahlawat et al. Supplementary Material

Supplementary Material

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Ahlawat et al. Supplementary Material

Supplementary Material

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