In experiment I, we detected pathological changes in the basal forebrain and hippocampus in 6- and 8-month-old APP/PS1 mice. The WT and AD mice groups were anesthetized with 1% pentobarbital sodium (0.05 g/kg i.p.). Saline and 4% paraformaldehyde were perfused from the left ventricle to, respectively, wash and fix the brain tissue. Then coronal slices of the brain tissue were prepared for pathological staining, using 5μm per section. To perform Thioflavin S staining, the tissues were placed in 0.3% Thioflavin S solution (Thioflavin S, Sigma, T1892) and then differentiated in 50% ethanol. Nissl staining was performed with Nissl Staining Solution (Nissl Staining Solution (Cresyl Violet), Solarbio, G3410) according to the manufacturer’s instructions.
Thioflavin s solution
Manufactured by Merck Group
Sourced in Germany, United States
Thioflavin S solution is a laboratory reagent used for the detection and visualization of amyloid fibrils. It is a fluorescent dye that binds specifically to beta-sheet structures in proteins, resulting in a characteristic yellowish-green fluorescence when exposed to ultraviolet light.
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Lab products found in correlation
Nissl staining solution cresyl violet, by Solarbio (1 mentions)
Thioflavin s, by Merck Group (1 mentions)
Zen 3, by Zeiss (1 mentions)
Congo red solution, by Merck Group (1 mentions)
Axio scope a1 microscope, by Zeiss (1 mentions)
Sudan black b solution, by Merck Group (1 mentions)
Fv10 asw 3.0 viewer, by Olympus (1 mentions)
Fv1000 confocal laser scanning microscope, by Olympus (1 mentions)
Probeon plus microscope slides, by Thermo Fisher Scientific (1 mentions)
Fluoroshield with dapi, by Merck Group (1 mentions)
Normal goat serum (ngs), by Agilent Technologies (1 mentions)
Fluorescence mounting medium, by Agilent Technologies (1 mentions)
Pen membrane slides, by Thermo Fisher Scientific (1 mentions)
16 protocols using thioflavin s solution
1
Pathological Changes in APP/PS1 Mice
2
Histological Detection of Amyloid Deposits
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Thioflavin S and Congo Red are two major histological stains used to detect any form of amyloid. The stainings were performed according to the protocol used for routine clinical diagnostics at the Department of Neuropathology at University Hospital Aachen, Germany. For Thioflavin S staining, hydrated paraffin sections were stained for 5 min in Mayer′s hemalum solution, washed with water for 5 min and then stained with 1% Thioflavin S solution (w/v in ddH2O; Sigma, Munich, Germany) for 5 min. Staining was differentiated in 70% ethanol, rinsed in ddH2O and mounted in glycerol-gelatin. Thioflavin S bound to amyloid emits green fluorescence.
For Congo Red staining, hydrated paraffin sections were stained for 10 min in Mayer′s hemalum solution, washed with water for 10 min, incubated in fresh 1% NaOH solution (w/v in 80% ethanol, Merck, Darmstadt, Germany) for 20 min and stained with 0.5% Congo Red solution (w/v in 80% ethanol/0.1% NaOH, Merck, Darmstadt, Germany) for 20 min. Staining was differentiated in 100% ethanol, and mounted in vitroclud. Histological sections were viewed and imaged with an Axio Scope.A1 microscope (Zeiss, Oberkochen, Germany) using ZEN 3.1 software (Zeiss, Oberkochen, Germany).
For Congo Red staining, hydrated paraffin sections were stained for 10 min in Mayer′s hemalum solution, washed with water for 10 min, incubated in fresh 1% NaOH solution (w/v in 80% ethanol, Merck, Darmstadt, Germany) for 20 min and stained with 0.5% Congo Red solution (w/v in 80% ethanol/0.1% NaOH, Merck, Darmstadt, Germany) for 20 min. Staining was differentiated in 100% ethanol, and mounted in vitroclud. Histological sections were viewed and imaged with an Axio Scope.A1 microscope (Zeiss, Oberkochen, Germany) using ZEN 3.1 software (Zeiss, Oberkochen, Germany).
3
Thioflavin S Staining of Amyloid Deposits
4
Tau Fibril Staining with Thioflavin S
5
Thioflavin S Staining of Murine Brain
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Mice were anesthetized with 5% chloral hydrate, and saline solution was used for perfusion through the heart, which was followed by 4% paraformaldehyde. The brains of the mice were then removed, fixed in 4% paraformaldehyde for 24 h and immersed in 30% sucrose until they sank. Next, 30-µm coronal floating sections were immersed in 1% Thioflavin S solution (Sigma-Aldrich; Merck, Darmstadt, Germany) for 9 min and differentiated in 70% alcohol for 5 min. Subsequently, sections were washed in 80 and 95% ethanol, followed by three washes with ddH2O, and were then mounted. Fluorescent signals were detected by fluorescence microscopy (Olympus BX51; Olympus Corp., Tokyo, Japan).
6
Thioflavin S Staining of NeuN
7
Amyloid-Beta Labeling in Mouse Brain
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Six brain sections at 210–240-μm intervals were extracted from each mouse from the region between −2.6 mm and −4.3 mm to the bregma with reference to Paxinos and Franklin’s the Mouse Brain in Stereotaxic Coordinates [93 ]. To label Aβ, the stored brain sections were washed three times for five minutes in PBS. The sections were incubated in filtered thioflavin-S solution (1%; Sigma-Aldrich Corporation, St. Louis, MO, USA) for 15 minutes and washed in 80% and 70% ethanol for one minute each. After washing three times for five minutes each in PBS, the sections were mounted on ProbeOn™ Plus Microscope Slides (Thermo Fisher Scientific Inc., Waltham, MA, USA) and coverslipped with the Fluoroshield™ with DAPI (Sigma-Aldrich Corporation, St. Louis, MO, USA).
8
Amyloid Plaque Visualization Protocol
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A 300-μm spaced series of three blind-coded 30 μm sections were fixed in 4% PFA for 10 min, followed by incubation in 0.25% potassium permanganate solution for 15 min, and a bleaching step with 1% potassium metabisulfite/1% oxalic acid for 5 min, incubation with 0.02% Thioflavin-S solution in 50% ethanol (T1892; Sigma-Aldrich) for 8 min, rinsed with water between every step. Finally the sections were incubated with 1 μl/ml DAPI. The sections were analyzed as described for the immunostained sections. Stained sections were scanned with the TissueFAXs microscope using 20X objective lens. Images were processed using Tissuequest software, selecting the cortex for automated analyses.
9
Immunofluorescent Staining of Tissue
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A second series incubated in anti-NeuN (1:1000), overnight, was followed by anti-rabbit-alexa-594 (1:500) for 5 h. Sections were rinsed then immersed in a 1% Thioflavin S solution (Sigma) for 9 min, rinsed in dH2O, destained in 70% Ethanol for 5 min, rinsed in dH2O, and then transferred to TBS before mounting onto slides.
10
Thioflavin-S Staining for Amyloid Detection
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For thioflavin-S staining, the slides were deparrafinized and hydrated with xylene and serial alcohol solutions. Stains were performed with 1% thioflavin-S solution (Sigma-Aldrich) for 10 min, and then the slides were washed in running tap water for at least 10 min. The slides were then dehydrated with alcohol, cleared in xylene, and mounted with mounting medium.
Thioflavin-S staining was quantified both automatically, involving the calculation of the positive staining area relative to the entire brain, and manually, by counting the number of positive spots in the cortex and hippocampus. These assessments were conducted in a blinded fashion. Additionally, we engaged a veterinarian pathologist in identifying true positive signals to perform these assessments in a blinded manner.
Thioflavin-S staining was quantified both automatically, involving the calculation of the positive staining area relative to the entire brain, and manually, by counting the number of positive spots in the cortex and hippocampus. These assessments were conducted in a blinded fashion. Additionally, we engaged a veterinarian pathologist in identifying true positive signals to perform these assessments in a blinded manner.
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