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NeuroScience Associates

Alzheimer Disease (AD)

See Table of Stains appropriate for AD

 

Overview

The hallmark pathological features of Alzheimer Disease (AD) in humans are amyloid plaques and neurofibrillary tangles. Over the past decade, an increasing number of genetically engineered mouse models for AD research have been developed to exhibit classic Alzheimer features. The timeline for development of disease features ranges from a few months to ~18 months. NSA has processed hundreds of human AD tissues and thousands of brains from AD mouse models.

Mouse Tissue:

When processing mouse brain hemispheres coronally, NSA embeds up to 40 in each MultiBrain®; when processing hemisphere or intact brains sagittally, up to 20 entire brains are embedded (25 if the area of interest is the cortex/cerebrum). The preference of the researcher determines which alignment is chosen.


Mouse Brain Hemisphere Coronal

40 mouse brain hemispheres are
co-embedded and appear on each
MultiBrain® slide section.


Mouse Brains Sagittal

20 mouse brains are co-embedded
and appear on each MultiBrain® slide
section (25 mouse brains if the area of
interest is only cortex/cerebrum).


Human Tissue:

With human tissue, NSA is capable of processing human brain and other large format sections, as in the image below, providing a unique opportunity to assess large contiguous cross-sections of tissue. More often however, NSA is asked to process multiple smaller samples from one or more brains using MultiBrain® Technology.

The standard NSA practice of encasing the brain tissue in gelatin provides a significant aid in the handling of tissue sections resulting in an improved final product.

Human Brain Hemisphere


Multiple Embedded Human Brain Tissues


Amyloid Plaques

The hallmark features of AD are amyloid plaques and tau abnormalities. Amyloid-laden plaques exist as a diffuse form and as a more dense/mature form (congophilic). Amyloid is also found as deposits in vessels. Tau abnormalities are present in cell bodies and in the neuropil as “neuropil threads.”

The choice of which stain(s) to use is based on the specific needs of the study. The Campbell-Switzer Alzheimer pathology stain, developed by NSA, shows all of the hallmark features of Alzheimer pathology, while other antibody methods reveal limited, specific features. Please note that the Campbell-Switzer stain does not show this pathology on monkey tissue.

Strategic Approach to Amyloid Detection: Different approaches yield unique features At specific times during an R&D cycle, different approaches to amyloid detection may be most useful. Each of the methods described has its own strengths and specialized advantage.

Campbell-Switzer MethodDr. Bob Switzer and his former associate, Shannon Campbell, developed the Campbell-Switzer Alzheimer Pathology stain in the early 1980’s. Over the years, this unique stain has become a “work horse” tool for researchers, particularly in the earlier phases of R&D. Diffuse plaques are stained black and the denser fibrillar amyloid forms are stained amber. Tau abnormalities typically stain black.

Immunohistochemistry for Amyloid Plaque Detection: The antibody methods are intended to be very specific and reveal amyloid peptides that have been cleaved at a specific location. On the basis of a particular mouse model, treatment approach and other factors, a specific antibody can be more useful than others in revealing a specific measure of efficacy.

Congo RedCongo Red specifically reveals the denser, fibrillar amyloid plaques (hence, congophilic plaques). While the fibrillar amyloid cores stain red, other entities in the sections can also be stained. To distinguish amyloid staining from other staining, the sections can be viewed with polarized microscopy.  Fibrillar amyloid stained with Congo Red is birefringent, so when viewed with crossed polarizers a ‘cross’ will be observed with one arm being red and the other ‘apple green’—often termed a ‘maltese cross’. Paired helical filaments that form the tangles in neurons will also display this phenomenon.  The birefringence occurs due to the congo red molecules binding in an orderly way on the beta pleated sheet-like configuration of the fibrillar amyloid and the paired helical filaments.

Thioflavin SThis stain, when bound to beta pleated sheet-like configurations of the amyloid and paired helical filaments, will fluoresce a yellow-green. This stain is a useful option if double staining with fluorescent markers are being used.

The following images were acquired by applying five common techniques to adjacently cut sections of the same Tg2576 mouse brain:

Congo Red Stain

Congo Red and Thioflavin S reveal only fibrillar amyloid (dense-core, congophilic) plaques. If the goal is to detect ONLY congophilic plaques, Congo Red and Thioflavin S are good staining choices.

Thioflavin S Stain

Aβ 1-40 IHC

Aβ 1-40 IHC displays a broader range of amyloid than the congophilic markers shown above. Aβ 1-42 IHC displays a broader range of features than Aβ 1-40.

Aβ 1-42 IHC


Campbell-Switzer Alzheimer
Pathology Stain


Alternatively, the Campbell-Switzer method stains the broadest range of amyloid while allowing for differentiation between congophilic and diffuse plaques: The Campbell-Switzer method appears to reveal a similar level of amyloid as Aβ 1-42 IHC. However, as can be seen in the close-up, the congophilic plaques stain a different color (amber) vs. the diffuse plaques (black), whereas with the Aβ 1-42 IHC method, both diffuse and congophilic appear the same color. The high-contrast images resulting from this stain are ideally suited for densitometric analysis of plaque loads.

Amyloid Plaques and Tau Abnormalities

6E10 Mouse Alzheimer Model


Aβ 1-42 Human Alzheimer

Early-onset, familial Alzheimer disease in
temporal lobe cortex

AB 1-28 IHC

The pathology is characterized by few neurons with tangles (black circular to tear-drop shapes) in hippocampus (right) while abundant in temporal lobe.

Blue = Nissl substance (RNA) in cell bodies of neurons and glia.
Black = Tau abnormalities and neuropil threads; Gallyas silver method.


Aβ 1-28 IHC images courtesy of Dr. Alex Osmond, University of Tennessee, Knoxville

Neuritic plaques (amber-brown) revealed by Aβ 1-28 immunohistochemistry in the hippocampus

Campbell-Switzer Alzheimer Pathology Stain (Human)Neuritic Plaques and Tau abnormalities in Striatum

Tau Abnormalities

Anti-Tau HT7 (Epitope 158-163)
Mouse P301L AD Model

Anti-Tau HT7 (Epitope 158-163)
Mouse P301L AD Model, 10x

AT8 (pSer202 + Thr205)
Surface Cells, P301L Mouse Cortex, 20x

AT8 (pSer202 + Thr205)
Unspecified AD Mouse Model, Cortex, 20x

AT8 (pSer202 + Thr205)
Unspecified AD Mouse Model, Hippocampus, 20x

AT8 (pSer202 + Thr205)
Unspecified AD Mouse Model, Hippocampus, 40x

Anti-Human Tau (Epitope 243-441)
Human AD Cortex, 20x

Anti-Human Tau (Epitope 243-441)
Magnified to show neuritic plaque and Tau
abnormalities, 40x

Tau 46 (Epitope 404-441)
Human AD Cortex, 10x

Tau 46 (Epitope 404-441)
Human AD Cortex, Magnified to show neuritic plaque
and Tau abnormalities, 20x

Tau 46 (Epitope 404-441)
Mouse P301L AD Model, Hemisphere

Tau 46 (Epitope 404-441)
Mouse P301L AD Model, Hippocampus

Tau pSer396
P301L Mouse Model, Cortex and Hippocampus, 10x

Tau pSer396
P301L Mouse Model, Hippocampus, 40x

Tau pSer422
P301L Mouse Model, Cortex and Hippocampus, 2x

Tau pSer422
P301L Mouse Model, Cortex and Hippocampus, 4x

Tau pSer422
Human Cortex, 10x

Tau pSer422
Human AD Cortex, 20x

Tau pThr181
Human AD Cortex, 1x

Tau pThr181
Human Cortex, 10x

Gallyas Silver Method for revealing Tau abnormalities
Human AD Hippocampus, 2x

Gallyas Silver Method
Human AD Hippocampus, 10x

Gallyas Silver Method
Human AD Hippocampus, 20x

Gallyas Silver Method
Mouse Cortex, 10x

Gallyas Silver Method
Mouse Cortex, 20x

Oligomers

Amyloid Oligomer A11
Human AD Cortex, 2x

Amyloid Oligomer A11
Human AD Cortex, 40x

Amyloid Oligomer A11
Human AD Dentate-Gyrus, 4x

Amyloid Oligomer A11
Human AD Dentate-Gyrus, 40x

Tau Oligomers

Anti-Tau T22 Oligomers, Human AD Cortex, 2x

Anti-Tau T22 Tau Oligomers in Human AD Cortex shown at
higher magnification in neuron cell bodies and dendrites, 20x

Anti-Tau T22 Tau Oligomers display neurites and tau abnormalities,
Human AD Cortex, 10x

Inflammation

Inflammation occurs in conjunction with the hallmark pathologic features of AD and can be detected by examining microglia (Iba1 IHC) or astrocytes (GFAP IHC).

Iba1 Immunoreactive Microglia: Alzheimer Disease Mouse Model

Campbell-Switzer Stain

Alzheimer Mouse Model Plaque Chemoarchitecture

Microglia-Iba1 Antibody Stain

The commonly high density of plaques found in the subiculum is shown. Co-located with the plaques, shown by the Campbell-Switzer method, are reactive microglia (Iba1 immunohistochemistry). The reactive microglia are more evenly distributed, rather than clustered tightly around particular plaques.

GFAP Immunoreactive Hypertrophic Astrocytes around Neuritic Plaque

 

Disintegrative Debris

Campbell-Switzer Alzheimer Stain

Sections of the brain of an old, >12 months, PS-1/APP mouse

AmCuAg Disintegrative Degeneration Stain

Numerous diffuse plaques (in black) and the fewer “mature” plaques (in amber) that contain fibrillar amyloid (congophilic, Thioflavin S positive) are depicted.

The degeneration stain above reveals degenerative globular profiles at the perimeter of the mature (amber) plaques but not with the diffuse plaques. These profiles have been identified as dystrophic neurites (Brendza et al. J Comp Neurol 456: 375–383, 2003). These may also be engorged glia processes.

AmCuAg Disintegrative Degeneration Stain
Reveals Axon Degeneration in APP mouse
Degenerated Axons Amidst Mature Plaques in Olfactory Peduncle

Degenerated Axons in the Corpus Callosum
and Striatal Fiber Bundles

Degenerated Axons in Optic Tract

Increase in Iron

 

Ferritin Immunoreactivity in a Mouse Model of Alzheimer Disease

Immunoreactivity of an antibody against ferritin (Sigma) in the cortex of a mouse. Ferritin synthesis is triggered by the presence of iron and may act as a persistent indicator of earlier as well as current levels of iron.
Staining with Perls-DAB for ferric iron does not yield the same image.

AD Human Cortex: Aβ 1-42 (brown)
Perls Stain for Ferric Iron (blue)

If you would like further information about how NSA can assist you with Alzheimers Disease research, please contact us or refer to our catalog.