AAT Bioquest

Neurodegeneration & Amyloid Staining

Staining of Thioflavin S
The staining of Thioflavin S (in green) on adjacent sections of the hippocampus of a patient suffering from Alzheimer's disease. Thioflavin S binds both senile plaques (SP) and neurofibrillary tangles (NFT), the two characteristic cortical lesions of Alzheimer's.
While their clinical manifestations vary, the protein-level mechanisms of amyloid related diseases appear to be incredibly similar. In amyloid related diseases, aberrant monomeric proteins undergo conformational shifts, facilitating the aggregation and formation of amyloid bodies. The protein dynamics of amyloid formation includes nucleation plus the growth of amyloid-like proteins. Additionally, interactions between the amyloid fibrils and native proteins can alter cellular interactions. The resulting cascade likely contributes to the late onset and accelerating progression of amyloid related disorders and the widespread effects that they have on the body. Such amyloid related disorders are generally termed amyloidosis, which is categorized by the accumulation of pathogenic fibrous amyloid bodies that do not have a role in structure, support, or motility.

Some well-studied forms of amyloidosis include Alzheimer's disease, spongiform encephalopathies (Mad cow disease), and type II diabetes, all of which are progressive and associated with high mortality and morbidity. Amyloidosis is often related to neurodegeneration, which is the degeneration of the brain and sometimes body, which gradually intensifies until incapacitation and death.



Amyloid Fibrils and Amyloid Bodies

Amyloid fibrils are homopolymers which are polymers made of identical monomer units. Amyloid fibrils encompass a broad range of polymers which may exhibit different protofilament numbers, arrangements, and conformation. It has been well documented that amyloid fibrils may assemble to form insoluble structures, resistant to degradation. These structures (also termed amyloid, amyloid bodies or deposits) exist as abnormal fibrous, extracellular deposits found in various organs and tissues. Though research interrogating the basis of amyloid structure is still evolving, it has been inferred that all types of amyloid consist of one major fibrillar protein, and all are structurally dominated by β-sheet conformation.

Research has shown that polymorphisms set the stage for abnormal protein folding in amyloid fibril deposition; amyloid formation thus involves protein misfolding reactions. Though amyloid bodies are composed of misfolded native proteins, they are not easily recognized or removed as a foreign substance by the immune system, for reasons that are not well understood.

In some microorganisms, specific proteins and domains may have evolved for making amyloid fibrils that play an important, essential role within the cell. In most cases, however, amyloid deposits are pathogenic, formed unintentionally under conditions of stress, from protein misfolding or misregulation. It has been shown that some amyloid deposits are directly involved in disease mechanisms, whereas other mechanistic roles of amyloid bodies are less clear. Certain proteins tend to associate with amyloid related diseases and are those that can create, or interact with, amyloid fibrils.

Some healthy functional proteins can also be induced to form amyloid fibrils through exposure to certain nonnative conditions in vitro. Due to the current association of amyloid bodies and various diseases, specifically those for neurodegeneration, it is thought that functional amyloid fibers may play a beneficial role in key physiologic processes like long-term memory formation and release of peptide hormones.

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Histopathological Features of Parkinson's and Alzheimer's Disease

Histopathological Features of Parkinson's and Alzheimer's Disease. From left to right: plaque accumulation due to α-synuclein and β-aggregates, neuron loss and degeneration, and neuroinflammation due to blood-brain-barrier (BBB) leakage. Figure made in BioRender.

Alzheimer's disease is the most prevalent form of dementia, impacting an estimated 6.7 million Americans aged 65 and older in 2023 alone. As Alzheimer's disease causes large-scale loss of synapses and neurons, particularly in regions of the brain associated with memory, neurodegeneration manifests as severe memory loss and loss of cognition.

In research, there have been two associated amyloid proteins involved in pathogenesis, the first of which is amyloid β (Aβ) involved in synaptic plasticity, learning, and memory, expressed ubiquitously in neurons. The second protein is Tau, which is involved in the stabilization of microtubules for intracellular transport. In the pathological form, amyloid β aggregates as plaque and misslocalizes outside of neurons into the mitochondria and soma. Similarly, the pathological form of Tau creates hyperphosphorylated neurofibrillary tangles of amyloid proteins and mislocalized outside of microtubules into dendrites and dendritic spines.

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Parkinson's disease is another amyloid related disease that presents a neuromotor disorder resulting from selective, progressive degeneration of dopamine-producing neurons. Parkinson's disease exhibits as progressive loss of conscious muscle control and results in trembling, stiffness, and/or retarded movement. There are two common amyloid structures associated with Parkinson's disease, including Lewy bodies and Lewy neurites that are located in the cell body and neurons, respectively. Commonly, the presynaptic protein α-synuclein has been found to aggregate in both.

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Other amyloid related disorders include Huntington's disease which causes selective neuronal death, primarily in the cortex and striatum of the brain. Huntington's disease commonly leads to motor disturbance, cognitive loss, and/or psychiatric issues. Huntington's disease has been linked to an inheritance driven mutation in the Huntingtin protein (Htt). In this mutation, the repeated expansion of a CAG trinucleotide repeats leads to the expansion of the polyglutamine (polyQ) tract and consequent formation of amyloid-like protein aggregates. This Htt mutation, though not quite fully understood, is thought to initiate from a disruption in nuclear transport, which leads to the accumulation of Htt in the nuclei of affected cells.

Amyotrophic lateral sclerosis (ALS) is a fourth amyloid related disorder that impacts neuromotor skills and is characterized by progressive muscle degeneration. ALS usually results in a loss of voluntary muscle control, and often leads to death from respiratory failure. There are two major subcategories of ALS that are either related to inheritance or not (familial ALS and sporadic ALS, respectively), and both are characterized by certain genetic mutations. These genes produce specific proteins, including superoxide dismutase 1 (SOD1), fused in sarcoma (FUS), TAR DNA binding protein 43 (TDP-43), and chromosome 9 open reading frame 72 (C9orf72), that all play central roles within the nucleus. In the pathological form, these proteins will mislocalize into the cytoplasm, rather in the nucleus, to form amyloid-like bodies.


Amyloid Staining Methods

Congo Red structure
Chemical structure for Congo Red *UltraPure grade* *CAS 573-58-0*
in vivo, amyloid deposits have been distinguished by some common characteristics. They present a fibrillar appearance on electron micrography and show an amorphous eosinophilic appearance on hematoxylin and eosin staining. Amyloids exhibit a β-pleated sheet structure, and an apple-green birefringence (double image effect) is seen in Congo red histological staining, which indicates a crystalline-like ordered structure. Notably, the prevalence of the β-pleated sheet conformation has been exploited to identify the presence of amyloid deposits; for this reason, Congo red is most commonly used to identify amyloid deposits. To a lesser extent other stains have been used in the literature for detecting amyloid bodies, including thioflavin S, thioflavin T, crystal violet, methyl violet, sirius red, periodic acid-Schiff (PAS), and toluidine blue.

Often, Congo red is the dye of choice because amyloids cause a spectral shift in the peak wavelength of the stain with conventional light microscopy. Amyloid deposits also show heightened intensity of fluorescence with Congo red, which suggests an increased capability of the stain to pack onto the substrate. It is well documented that Congo red shows an apple-green birefringence when viewed by polarizing microscopy, though fluorescence microscopy may be the optimal form of visualization since this method can easily detect small and/or weakly stained amyloid deposits. Importantly, Congo red does not significantly stain any other tissue components other than amyloid, outside of elastic fibers and coarse collagen fibers. Typically, additional stains are included in the procedure to quench background fluorescence and facilitate viewing.
The efficiency of Congo red to detect amyloid bodies comes from its structure; Congo red contains linear dye molecules that deposit onto the long axis of amyloid fibrils. Congo red binds ionically with proteins via two sulfonic acid groups, which results in a tightly packed series of dye molecules over the entire length of the fibril.

Along with the histological stain, a second step in experimentation is usually included for the identification of the amyloidogenic protein. Various approaches for amyloid typing are currently used: commonly, immunofluorescence on fresh-frozen cryostat sections; immunohistochemistry on fixed paraffin-embedded tissues; immunogold labeling on transmission electron microscopy ultrathin sections; or laser microdissection followed by mass spectrometry-based proteomics. Though laser microdissection and mass spectrometry may be the most precise method for identifying amyloid related proteins, immunohistochemistry is often the go-to for its ease, commercial availability, inexpensiveness, quick testing times, and sensitive and reliable results.



Example Staining Procedure

  1. Prepare a saturated Congo red solution by adding 2.5 g of Congo Red to a total volume of 250 ml of 50% ethyl alcohol. Histological sections that are already mounted on slides work best, which should be defatted in xylene or similar.
  2. Hydrate the slides through multiple ethyl alcohol solutions (100%, 95%, 80% and 70%, 1-2 min each).
  3. Stain the slides in the Congo red solution for 1 hr. After the time has elapsed, carefully dip the slides into a saturated lithium carbonate solution for 10-20 sec and rinse them for 10-20 sec in distilled or deionized water.
  4. Dip the slides in 70% ethanol for 5-60 sec to prevent background staining. Once complete, transfer slides through ethanol solutions (80%, 95%, and twice with 100%, 15 sec - 2 min each).
  5. Slides should then be placed in xylene or similar for 5-10 min. Slides can then be cover-slipped with an appropriate mounting media and viewed in fluorescence or plane-polarized light.
Note: It is important to note that even though much research has exclaimed an “apple-green birefringence” of Congo red-stained amyloid deposits in polarized light, this is all dependent on microscopy conditions. Both the polarizer and analyzer in the microscope can impact the color, and Congo red-stained amyloid deposits have been visualized as red, blue, green, yellow, and even transparent under different conditions (Howie, et al., 2018).


Product Ordering Information

Table 2. Neuronal Tracers

Product Name
Ex/Em (nm)
Standard Filter Set
Chroma Filter
Omega Filter
Unit Size
22038DiOC2(3) iodide [3,3-Diethyloxacarbocyanine iodide]482/500FITC31001-ChromaXF23-Omega25 mg
22039DiOC3(3) iodide [3,3-Dipropyloxacarbocyanine iodide]482/500FITC31001-ChromaXF23-Omega25 mg
22040DiOC7(3) iodide [3,3-Diheptyloxacarbocyanine iodide]482/500FITC31001-ChromaXF23-Omega25 mg
22042DiOC16(3) perchlorate [3,3-Dihexadecyloxacarbocyanine perchlorate]482/500FITC31001-ChromaXF23-Omega25 mg
22045DiOC5(3) iodide [3,3-Dipentyloxacarbocyanine iodide]482/500FITC31001-ChromaXF23-Omega25 mg
22046DiOC6(3) iodide [3,3-Dihexyloxacarbocyanine iodide]482/500FITC31001-ChromaXF23-Omega25 mg
22066DiO perchlorate [3,3-Dioctadecyloxacarbocyanine perchlorate]482/500FITC31001-ChromaXF23-Omega25 mg
21483MM 1-43 [N-(3-Triethylammoniumpropyl)-4-(4-(dibutylamino)styryl)pyridinium dibromide]473/579TRITC31002-ChromaXF32-Omega1 mg
22035DiIC12(3) perchlorate [1,1-Didodecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate]549/563TRITC31002-ChromaXF32-Omega25 mg
22044DiIC16(3) perchlorate [1,1-Dihexadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate]549/563TRITC31002-ChromaXF32-Omega25 mg
22050DiIC12(3)-DS [1,1-Diododecyl-3,3,3,3-tetramethylindocarbocyanine-5,5-disulfonic acid]549/563TRITC31002-ChromaXF32-Omega5 mg
22052DiIC18(3)-DS [1,1-Dioctadecyl-3,3,3,3-tetramethylindocarbocyanine-5,5-disulfonic acid]549/563TRITC31002-ChromaXF32-Omega5 mg
22073DiSC2(3) [3,3-Diethylthiacarbocyanine iodide]560/571TRITC31002-ChromaXF32-Omega25 mg
22101DiI iodide [1,1-Dioctadecyl-3,3,3,3- tetramethylindocarbocyanine iodide]549/563TRITC31002-ChromaXF32-Omega100 mg
22102DiI perchlorate [1,1-Dioctadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate] *CAS 41085-99-8*549/563TRITC31002-ChromaXF32-Omega100 mg
22103DiI triflate [1,1-Dioctadecyl-3,3,3,3-tetramethylindocarbocyanine triflate]549/563TRITC31002-ChromaXF32-Omega100 mg
21489MM 2-10 [N-(3-Triethylammoniumpropyl)-4-(4-(diethylamino)styryl)pyridinium dibromide]506/620Texas Red31024-ChromaXF21-Omega1 mg
22030DiA [4-(4-(Dihexadecylamino)styryl)-N-methylpyridinium iodide]491/611Texas Red31024-ChromaXF21-Omega25 mg
22033DiD labeling solution [1,1-Dioctadecyl-3,3,3,3-tetramethylindodicarbocyanine] *5 mM DMSO solution*645/663Cy531023-ChromaXF47-Omega10 mL
22051DiIC12(5)-DS [1,1-Diododecyl-3,3,3,3-tetramethylindodicarbocyanine-5,5-disulfonic acid]650/670Cy531023-ChromaXF47-Omega5 mg
22054DiIC18(5)-DS [1,1-Dioctadecyl-3,3,3,3-tetramethylindodicarbocyanine-5,5-disulfonic acid]652/668Cy531023-ChromaXF47-Omega5 mg
22056DiIC1(5) iodide [1,1,3,3,3,3-Hexamethylindodicarbocyanine iodide]640/657Cy531023-ChromaXF47-Omega25 mg
22076DiSC3(5) [3,3-Dipropylthiadicarbocyanine iodide]660/675Cy531023-ChromaXF47-Omega25 mg
21487MM 4-64 [N-(3-Triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridinium dibromide]558/734Cy5.5--1 mg
22070DiR iodide [1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyanine iodide]754/778Cy741009-ChromaXF112-Omega25 mg
3080Biocytin *CAS 576-19-2* 100 mg
3085Biocytin C2 maleimide 5 mg

Table 3. Tau Antibodies



Amyloid fibrils
Alzheimer's Disease Facts and Figures
The role of amyloid oligomers in neurodegenerative pathologies
Histological Staining of Amyloid and Pre-Amyloid Peptides and Proteins in Mouse Tissue
Amyloid from a histochemical perspective. A review of the structure, properties and types of amyloid, and a proposed staining mechanism for Congo red staining
Physical basis of colors seen in Congo red-stained amyloid in polarized light
Immunohistochemical typing of amyloid in fixed paraffin-embedded samples by an automatic procedure: Comparison with immunofluorescence data on fresh-frozen tissue