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Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Neuroscience

Correlative Light and Electron Microscopy to Study Microglial Interactions with β-Amyloid Plaques

Published: June 1st, 2016

DOI:

10.3791/54060

1Neurosciences Axis, CHU de Québec Research Center
* These authors contributed equally

This article describes a protocol for visualizing amyloid Aβ plaques in Alzheimer's disease mouse models using methoxy-X04, which crosses the blood-brain barrier and selectively binds to β-pleated sheets found in dense core Aβ plaques. It allows pre-screening of plaque-containing tissue sections prior to immunostaining and processing for electron microscopy.

A detailed protocol is provided here to identify amyloid Aβ plaques in brain sections from Alzheimer's disease mouse models before pre-embedding immunostaining (specifically for ionized calcium-binding adapter molecule 1 (IBA1), a calcium binding protein expressed by microglia) and tissue processing for electron microscopy (EM). Methoxy-X04 is a fluorescent dye that crosses the blood-brain barrier and selectively binds to β-pleated sheets found in dense core Aβ plaques. Injection of the animals with methoxy-X04 prior to sacrifice and brain fixation allows pre-screening and selection of the plaque-containing brain sections for further processing with time-consuming manipulations. This is particularly helpful when studying early AD pathology within specific brain regions or layers that may contain very few plaques, present in only a small fraction of the sections. Post-mortem processing of tissue sections with Congo Red, Thioflavin S, and Thioflavin T (or even with methoxy-X04) can label β-pleated sheets, but requires extensive clearing with ethanol to remove excess dye and these procedures are incompatible with ultrastructural preservation. It would also be inefficient to perform labeling for Aβ (and other cellular markers such as IBA1) on all brain sections from the regions of interest, only to yield a small fraction containing Aβ plaques at the right location. Importantly, Aβ plaques are still visible after tissue processing for EM, allowing for a precise identification of the areas (generally down to a few square millimeters) to examine with the electron microscope.

Amyloid Aβ plaque formation is the main neuropathological hallmark of Alzheimer's disease (AD). However increasing evidence suggests important roles of the immune system in disease progression1,2. In particular, new data from preclinical and clinical studies established immune dysfunction as a main driver and contributor to AD pathology. With these findings, central and peripheral immune cells have emerged as promising therapeutic targets for AD3. The following protocol combines light and electron microscopy (EM) to generate new insights into the relationship between Aβ plaque deposition and microglial phenotypic alterations in AD. ....

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Note: All experiments were approved and performed under the guidelines of the Institutional animal ethics committee, in conformity with the Canadian Council on Animal Care guidelines as administered by the Animal Care Committee of Université Laval. APP-PS1 male mice between 4 and 21 months of age were used. These animals were housed under a 12 hr light-dark cycle at 22 - 25 °C with free access to food and water.

1. Methoxy-X04 Solution Preparation

  1. Prepare a 5 mg/ml s.......

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This section illustrates the results that can be obtained at different critical steps of the protocol. In particular the results show examples of brain sections containing methoxy-X04 stained plaques in specific region and layers of interest: the hippocampus CA1, strata radiatum, and lacunosum-moleculare. The plaques and regional/lamellar organization of the hippocampus are successively visualized using a combination of UV and bright field filters (Figure 1). The selected.......

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This protocol explains a correlative approach for targeting dense core Aβ plaques with EM. Methoxy-X04 in vivo injection allows rapid selection of brain sections that contain Aβ plaques within particular regions and layers of interest, for instance the hippocampus CA1, strata radiatum, and lacunosum-moleculare. In the present example, methoxy-X04 pre-screening was combined with pre-embedding immunostaining for IBA1 to study how different microglial phenotypes interact with synapses at the ultrastructur.......

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We are grateful to Dr. Sachiko Sato and Julie-Christine Lévesque at the Bioimaging Platform of the Centre de recherche du CHU de Québec for their technical assistance. Grants from the Natural Sciences and Engineering Research Council of Canada (NSERC) RGPIN-2014-05308, The Banting Research Foundation, and The Scottish Rite Charitable Foundation of Canada to M.E.T supported this work.

H.E.H. is recipient of a scholarship from the Lebanese Ministry of Education and Higher Education, and K.B. from the Faculté de médecine of Université Laval.

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Name Company Catalog Number Comments
Methoxy-X04 Tocris Bioscience 4920 10 mg substrate per tablet
Propylene glycol Sigma Aldrich W294004 
Dimethyl sulfoxide (DMSO) Fisher BioReagents BP231-1 Caution: toxic
Sodium Chloride NaCl Sigma S9625
Sodium phosphate monobasic monohydrate Sigma S9638
Sodium phosphate dibasic Sigma S0876
Tris Hydrochloride Fisher BioReagents BP153500
Acrolein  Sigma 110221 Caution: Toxic
Paraformaldehyde Granular Electron Microscopy Sciences 19210 Caution: Toxic
Filter paper Fisher 09-790-14F
Peristaltic Pump with Tubing ColeParmer cp.78023-00
Excel Winged blood Collection Set Needles 25G  Becton Dickinson 367341
Extrafine Forceps F.S.T 11152-10 Tip shape: curved
Scissors F.S.T 14090-09 Tip shape: straight
Hartman Hemostats F.S.T 13003-10 Tip shape: curved
Surgical Scissors F.S.T 14004-16 Tip shape: straight
Micro Dissecting Scissors ROBOZ surgical store 5818
Glass scintillation vials Fisher Scientific 74515-20
Vibrating Blade Michrotome Leica VT1000 S Leica Biosystems  14047235612
Vibratome blades Electron microscopy Sciences 71990
Microscope Slides Fisher Scientific 12-550-15
24-well Tissue Culture Plates Fisher Scientific 353047
Ethylene Glycol Fisher BioReagents BP230-4
Glycerol Fisher BioReagents BP229-4
Hydrogen Peroxide, 30% J.T.BAKER cat: 2186-01
Sodium borohydride Sigma 480886
Tris HCl Fisher BP153-500ML
Fetal bovine Serum (FBS) Sigma Aldrich F1051
Bovine serum albumin (BSA), fraction V Thomas Scientific C001H24
Triton X-100 Sigma T8787
Anti IBA1, Rabbit  WAKO 019-19741
Goat Anti-Rabbit IgG Jackson Immunoresearch 111066046
VECTASTAIN Elite ABC Kit (Standard) Vector Labs PK-6100
3.3'-Diaminobenzidine tetra-hydrochloride (DAB) Sigma D5905-50TAB Caution: toxic
Osmium tetroxide, 4% solution Electron Microscopy Sciences 19150 Caution: toxic
Durcupan™ ACM single component A Sigma 44611 Resin Caution: Toxic
Durcupan™ ACM single component B Sigma 44612 Hardener Caution: Toxic
Durcupan™ ACM single component C Sigma 44613 Plasticizer Caution: Toxic
Durcupan™ ACM single component D Sigma 44614 Accelerator Caution: Toxic
Ethanol LesAlcoolsdeComerce 151-01-15N
Propylene oxide Sigma 110205 Caution: corrosive
Aluminum weigh dishes Electron Microscopy Sciences 70048-01
ACLAR®–Fluoropolymer Films Electron Microscopy Sciences 50425
Oven/Incubator VWR

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