Abstract
The brain vasculature can be investigated in different ways ranging from in vivo to biochemical analysis. Immunohistochemistry is a simple and powerful technique that can also be applied to archival tissues. However, staining of brain vessels on paraffin sections has been challenging. In this study, we developed an optimized method that can be used in paraffin-embedded mouse and human brain tissues derived from healthy controls and neurological disorders such as Alzheimer’s disease. We subsequently showed that this method is fully compatible with the detection of glial cells and key markers of Alzheimer’s disease including amyloid beta and phosphorylated Tau protein. Furthermore, we observed that the length of microvasculature in hippocampus of TgCRND8 Alzheimer’s disease mouse model is reduced, which is correlated with the decreased blood flow in hippocampus as determined by arterial spin labeling perfusion magnetic resonance imaging. Finally, we determined that the microvasculature length in two other Alzheimer’s disease mouse models, APP and PS1 double-transgenic mice and P301S Tau-transgenic mice, is also shortened in the dentate gyrus. Thus, we have established a new, simple and robust method to characterize the brain vasculature in the mouse and human brain.
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Acknowledgements
This work was funded by Grants to K.F. and Y.L. from SNOWBALL, an EU Joint Programme for Neurodegenerative Disease (JPND) (01ED1617B). We thank Professor Frank Kirchhoff for providing us NG2-CreERT2 mice that were crossbred with Rosa26-tdTomato animals. We also thank Elisabeth Gluding, Karin Heintz and Mirjam Müller for excellent technical assistance.
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Yang Liu and Klaus Fassbender share the senior authorship.
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Supplementary Fig. 1. Automatized quantification of blood vessel length. The color channels are split and only the channel containing the vasculature is kept (a). The vessels are thresholded to obtain the maximum intensity of the vascular staining (b). A mask is created; vessels are now represented in black (c). The mask is skeletonized to allow automatized length calculation (d) (PDF 786 KB)
429_2017_1595_MOESM2_ESM.pdf
Supplementary Fig. 2. Citrate-pepsin combined antigen-retrieval method does not interfere with astrocyte markers and the tandem dimer tomato protein in paraffin sections. Illustration of co-staining with collagen IV and GFAP in AD mouse models (a) or in AD brain tissues (b). Example of co-staining with DyLight® 488 labeled tomato lectin and pericytes (c,d). Pericytes restricted to the vasculature were detected with an antibody against red fluorescent protein in NG2-CreERT2 mice that were crossbred with Rosa26-tdTomato animals. Scale bar 50 μm (PDF 399 KB)
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Decker, Y., Müller, A., Németh, E. et al. Analysis of the vasculature by immunohistochemistry in paraffin-embedded brains. Brain Struct Funct 223, 1001–1015 (2018). https://doi.org/10.1007/s00429-017-1595-8
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DOI: https://doi.org/10.1007/s00429-017-1595-8