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Thioflavine s

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Thioflavine-S is a fluorescent dye commonly used in laboratory settings. It functions as a stain for the detection and visualization of amyloid fibrils in biological samples.

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Axio observer a1, by Zeiss (3 mentions) Lsm 780, by Zeiss (2 mentions) Q color 5 digital camera, by Olympus (1 mentions) Everbrite mounting media, by Biotium (1 mentions) Trueblack lipofuscin autofluorescence quencher, by Biotium (1 mentions) Bx51 microscope, by Olympus (1 mentions) Optiphot 2 fluorescence microscope, by Nikon (1 mentions) Envision plus system, by Agilent Technologies (1 mentions) Psyn 64, by Fujifilm (1 mentions) Bz x800e, by Keyence (1 mentions) Phosphoramidon, by Merck Group (1 mentions) Ab2636, by Abcam (1 mentions) Ab3280, by Abcam (1 mentions) Chloroquine, by Merck Group (1 mentions) Mouse monoclonal anti ha, by Merck Group (1 mentions) Mouse monoclonal anti flag m2 antibody, by Merck Group (1 mentions) Leupeptin, by Merck Group (1 mentions) Lyso tracker red, by Beyotime (1 mentions) Fluorescent mounting medium, by Agilent Technologies (1 mentions) Alexa fluorophore conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)

21 protocols using thioflavine s

1

Histological Staining of Brain Tissue

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Coronal sections were collected in order, mounted on slides, dried overnight at room temperature, stained with cresyl violet, dehydrated, and coverslipped. For thioflavine-S staining, the free-floating sections stained in 0.1% thioflavine-S according to manufacturer’s protocol (Sigma) mounted in 80% glycerol at pH 4.0. Thiazin red (TR) staining was performed as previously described60 (link) using aqueous 0.001% TR (gift from Dr. Jose Luna-Munoz).
Di J., Cohen L.S., Corbo C.P., Phillips G.R., El Idrissi A, & Alonso A.D. (2016). Abnormal tau induces cognitive impairment through two different mechanisms: synaptic dysfunction and neuronal loss. Scientific Reports, 6, 20833.
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2

Immunohistochemical Analysis of Brain Tissue

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Paraformaldehyde-fixed brains were sectioned coronally (at 20 μm thickness), using a sliding microtome (CM1900; Lieca, Nussloch, Germany). For immunohistochemistry (IHC), slices were blocked with phosphate-buffered saline (PBS) containing 10% fetal bovine serum (FBS) and 0.5% triton X-100 for 1 hour, incubated overnight with anti-GFAP primary antibody (Z0334; Dako Cytomation, Glostrup, Denmark) and anti-synaptophysin antibody (04–1019, Millipore), at 4 °C, and then in DyLight 488-AffiniPure anti-rabbit IgG secondary antibody (111–485–003; Jackson ImmunoResearch, Westgrove, PA, USA) for 1 hour. For Thioflavine-S staining, slices were stained with 0.015% Thioflavine-S (T1892; Sigma, MO, USA) for 15 minutes at room temperature. After mounting, slides were imaged using a Zeiss fluorescence microscope (Axio Observer A1; Zeiss, Oberkochen, Germany).
Chang W.H., Chen M.C, & Cheng I.H. (2015). Antroquinonol Lowers Brain Amyloid-β Levels and Improves Spatial Learning and Memory in a Transgenic Mouse Model of Alzheimer’s Disease. Scientific Reports, 5, 15067.
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3

Histological Analyses of Choroid Plexus and Cortex

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The in situ hybridization (ISH) analysis, immunohistochemistry (IHC) analysis and immunofluorescence (IF) analysis were performed as we previously reported (Zeng et al., 2019). Negative control sections were processed by omitting the complementary RNA or primary antibody. Nissl staining (0.5% cresyl violet) and Congo red staining (0.5% Congo red) were performed the same as previous studies (Zeng et al., 2019). For Thioflavine‐S staining, the slides were incubated with 0.05% Thioflavine‐S (Sigma) and then differentiated in 80% ethanol solution. Each section containing one microscopic field of the choroid plexus was digitized using a 40× objective. The total area of CAA was analyzed as percentage of total cortex area, and seven different sections from each mouse were determined in CAA. For other histological evaluation, three microscopic fields in hippocampus or cortex of each section were examined, respectively. All slides were mounted and imaged on a Nikon microscope or ZEISS LSM800. IF analysis of cultured cells was performed in the same way as tissue sections. The primary antibodies we used were showed in Table 1.
Zhao Y., Zeng C., Li X., Yang T., Kuang X, & Du J. (2020). Klotho overexpression improves amyloid‐β clearance and cognition in the APP/PS1 mouse model of Alzheimer's disease. Aging Cell, 19(10), e13239.
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4

Thioflavine-S Amyloid Staining

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Deparaffinized sections were incubated with 1% thioflavine-S (Millipore Sigma, St. Louis, USA; Cat# 1326-12-1) for 5 min [25 (link)]. 70% ethanol was then used for 5 min to differentiate, followed by rinsing with distilled water. Brain sections were coverslipped with anti-fluorescent quencher.
Wang T., Chen Y., Zou Y., Pang Y., He X., Chen Y., Liu Y., Feng W., Zhang Y., Li Q., Shi J., Ding F., Marshall C., Gao J, & Xiao M. (2022). Locomotor Hyperactivity in the Early-Stage Alzheimer’s Disease-like Pathology of APP/PS1 Mice: Associated with Impaired Polarization of Astrocyte Aquaporin 4. Aging and Disease, 13(5), 1504-1522.
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5

Amyloid-beta Plaque Visualization

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thioflavine-S staining was used to detect Aβ plaques (arrowheads) in the hippocampus and adjacent cortex. Deparaffinized sections were incubated with 1% thioflavine-S (MilliporeSigma; Cat# 1326-12-1) for 5 minutes. Tissue was differentiated in 70% ethanol for 5 minutes, followed by rinsing with distilled water. The brain sections were then coverslipped with anti-fluorescent quencher.
Liu Y., Hu P.P., Zhai S., Feng W.X., Zhang R., Li Q., Marshall C., Xiao M, & Wu T. (2022). Aquaporin 4 deficiency eliminates the beneficial effects of voluntary exercise in a mouse model of Alzheimer's disease. Neural Regeneration Research, 17(9), 2079-2088.
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6

In Vitro Plaque and Vascular Binding of PiB

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To visually characterize plaque and vascular binding of PiB in vitro, we used fixed tissue sections from the frontal cortex of two control cases (19 years and 51.3 years), DS (19.8 years), and DS with AD (DSAD) case (51.4 years) and the fluorescent cyano-PiB (CN-PiB). Sections were first mounted on slides and allowed to dry prior to incubation in 100 nM CN-PiB for 1 hour at RT using a similar protocol as published previously (Ikonomovic, et al., 2008 (link)) but at a lower concentration closer to the CN-PiB Kd to focus on high affinity imaging ligand-relevant binding. Slides were washed in PBS for 3× for 2 minutes then incubated briefly (30s) in TrueBlack™ lipofuscin autofluorescence quencher (Biotium, Hayward, CA). After three more 2-minute washes in PBS, slides were coverslipped using Everbrite™ mounting media (Biotium). Images were captured using an Olympus BX51 microscope with a Q Color 5 digital camera. A second set of sections was first incubated in CN-PiB as described above but prior to Trueblack quenching for autofluorescence, slides were incubated in 0.5% thioflavine-S (Sigma-Aldrich, St. Louis, MO) in 50% ethanol, differentiated in 50% ethanol, washed, and then exposed to TrueBlack. Sections were coverslipped using Everbrite™.
LeVine H I.I.I., Spielmann H.P., Matveev S., Cauvi F.M., Murphy M.P., Beckett T.L., McCarty K., Lott I.T., Doran E., Schmitt F, & Head E. (2017). Down syndrome: Age-dependence of PiB binding in postmortem frontal cortex across the lifespan. Neurobiology of aging, 54, 163-169.
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7

Amyloid Plaque Identification via Thioflavin S

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Slides from a subset of subjects were labeled with thioflavine S to verify whether plaques contained amyloidogenic fibrils. Sections processed for thioflavine S were defatted in 1:1 chloroform and 100% ethanol for 2 h, rehydrated as above, and stained with 0.5% aqueous thioflavine S (Sigma-Aldrich) for 20 min at RT, followed by differentiation in 80% ethanol. After several dH2O rinses, slides were coverslipped with an aqueous mounting medium (Gel-Mount; Biomeda, Foster City, CA, USA). Thioflaving S was visualize with the aid of a Nikon Optiphot-2 fluorescence microscope.
Kelley C.M., Perez S.E, & Mufson E.J. (2019). Tau pathology in the medial temporal lobe of athletes with chronic traumatic encephalopathy: a chronic effects of neurotrauma consortium study. Acta Neuropathologica Communications, 7, 207.
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8

Amyloid Plaque Visualization

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Brains were dissected and processed for serial paraffin sections (6 µm). By conventional dewaxing and rehydration, sections were immersed for 20 min in 0.1% Thioflavine-S (Sigma-T1892, Sigma-Aldrich, United States) PBS solution at room temperature. The green fluorescence-stained plaques were visualized by fluorescence microscopy. The number of plaques in the cerebral cortex and hippocampus was analyzed respectively.
Tan Y., Wang X., Zhang J., Zhang H., Li H., Peng T., Chen W., Wei P., Liu Z., He F., Li J., Ding H., Li N., Wang Z., Zhang Z, & Hua Q. (2022). NeuroProtect, a Candidate Formula From Traditional Chinese Medicine, Attenuates Amyloid-β and Restores Synaptic Structures in APP/PS1 Transgenic Mice. Frontiers in Pharmacology, 13, 850175.
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9

Immunohistochemical Analysis of Neurodegenerative Markers

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The tissue was rehydrated in a graded series of xylene and alcohols and steamed with distilled water for antigen retrieval. Sections were stained for αsyn (4B12, Biolegend, San Diego, CA, USA; 5G4, Millipore, Burlington, MA, USA), phosphorylated-αsyn pS129 (pSyn#64, WAKO, Richmond, VA, USA), tau (E1, [kind gift from Dr. Len Petrucelli and Dr. Casey Cook]), phosphorylated-tau pS202, Thr205 (AT8, Invitrogen, Waltham, MA, USA), and amyloid plaque (Thioflavine S, Sigma-Aldrich, St. Louis, MO, USA) and were visualized using the Envision Plus system (DAKO, Carpinteria, CA, USA). Counterstaining was performed with hematoxylin and 1× Scott’s tap water. The slides were subsequently dehydrated in a graded series of alcohols and xylenes and were coverslipped. All slides were scanned with the Aperio AT2 brightfield scanner (Deer Park, IL, USA) with a magnification of 20×. Immunofluorescence was scanned utilizing the Keyence BZ-X800E (Itasca, IL, USA) with a magnification of 20×. These original scans were used for final quantification, and then, images were zoomed in to allow for a close-up visualization of protein labeling.
Lim M.J., Boschen S.L., Kurti A., Castanedes Casey M., Phillips V.R., Fryer J.D., Dickson D., Jansen-West K.R., Petrucelli L., Delenclos M, & McLean P.J. (2023). Investigating the Pathogenic Interplay of Alpha-Synuclein, Tau, and Amyloid Beta in Lewy Body Dementia: Insights from Viral-Mediated Overexpression in Transgenic Mouse Models. Biomedicines, 11(10), 2863.
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10

Quantifying Amyloid Plaque Load

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Coronal free-floating sections were incubated for 9 min in 1% Thioflavine S (Sigma-Aldrich) aqueous solution and then treated with 80% ethanol two times for 3 min each, followed by a wash with 95% ethanol for 3 min. Sections were rinsed three times with distilled water. Thioflavine S imaging was performed with a confocal Zeiss laser-scanning-microscope using Zen software (LSM 780, Zeiss, Jena, Germany). A 5× magnification objective was used and green fluorescence was excited with a 488 nm Argon laser. Primary images were converted to 8 bit gray-scale and after defining the region of interest (total hippocampal or cortical area) they were thresholded using NIH ImageJ software (https://imagej.nih.gov/ij/). Thioflavine S plaque load was expressed as the percentage area covered by Thioflavine S-positive staining (% ThioS). The ImageJ tool ‘Analyze particles’ was used to determine plaque number and size (plaque area in µm2). The quantification was performed blind to the treatment for WT and AD mice.
Kartalou G.I., Salgueiro-Pereira A.R., Endres T., Lesnikova A., Casarotto P., Pousinha P., Delanoe K., Edelmann E., Castrén E., Gottmann K., Marie H, & Lessmann V. (2020). Anti-Inflammatory Treatment with FTY720 Starting after Onset of Symptoms Reverses Synaptic Deficits in an AD Mouse Model. International Journal of Molecular Sciences, 21(23), 8957.
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