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

Summary

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.

Abstract

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.

Introduction

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 progression^1,2. 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. ....

Protocol

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 solution of methoxy-X04⁹ by dissolving methoxy-X04 into a solution containing 10% dimethyl sulfoxide (DMSO), 45% propylene glycol, and 45% s....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

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 25 G	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 Microtome 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