The fluorescent pigment Thioflavin-S (Th-S) (Merck) was used to detect the deposition of amyloid plaques. Th-S has a peak of excitation and maximum emission of 430 and 550 nm, respectively. Brain sections were incubated with 0.1% Th-S in 50% ethanol for 10 min. After two washes in 50% ethanol and one wash in distilled water, sections were mounted using Mowiol Mounting Medium. Images were acquired using an Axiovert200 inverted microscope (Zeiss) coupled to a sCMOS monochrome camera (Excitation 461–488 nm, Emission 499–530 nm) with a dry 2.5× objective and were analyzed with FIJI software.
Thioflavin s ths
Manufactured by Merck Group
Sourced in United States
Thioflavin S (ThS) is a fluorescent dye used in laboratory settings. It is a core component for the detection and quantification of amyloid fibrils.
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Lab products found in correlation
Axiocam mrc, by Zeiss (2 mentions)
Axiovert 200 inverted microscope, by Zeiss (1 mentions)
Axiovert 200, by Zeiss (1 mentions)
Axiovert 200m microscope, by Zeiss (1 mentions)
Fluorescence mounting media, by Agilent Technologies (1 mentions)
Phenoimager ht automated quantitative pathology imaging system, by Akoya Biosciences (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Stereo lumar v12 microscope, by Zeiss (1 mentions)
Pannoramic 250 flash 2, by 3DHISTECH (1 mentions)
Horse serum, by Thermo Fisher Scientific (1 mentions)
α synuclein, by BD (1 mentions)
Dm2500, by Leica camera (1 mentions)
Hoechst 33342, by Merck Group (1 mentions)
Paraformaldehyde (pfa), by Biosesang (1 mentions)
Pkh26 red fluorescent cell linker kit, by Merck Group (1 mentions)
Fluoromount g, by Electron Microscopy Sciences (1 mentions)
Alexa fluor 594 goat anti rabbit igg, by Cell Signaling Technology (1 mentions)
Rabbit polyclonal anti ngf, by Abcam (1 mentions)
Horse anti mouse, by Cell Signaling Technology (1 mentions)
Goat anti rabbit igg hrp linked antibody, by Cell Signaling Technology (1 mentions)
12 protocols using thioflavin s ths
1
Amyloid Plaque Detection by Thioflavin-S
2
Visualizing β-Amyloid Deposition in Brain Sections
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The deposition of β-amyloid was detected by the fluorescent pigment thioflavin S (Th-S) (Merck), whose peak of excitation and maximum emission are 430 and 550 nm, respectively. The fixed brain sections were incubated for 15 min with 0.1% Th-S in 50% diluted ethanol, after which they were washed 2 times with 50% ethanol and subsequently with distilled water. The images were acquired with an inverted fluorescence microscope Axiovert 200 (Zeiss) coupled to a sCMOS camera (Excitation 461–488 nm, Emission 499–530 nm).
3
Thioflavin S and Congo Red Staining of Zona Pellucida
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Thioflavin S (ThS) (Sigma Chemical Co., St. Louis, MO) was prepared as a 1% stock solution in PBS, filtered and stored in the dark. Isolated ZPs were transferred to 0.1% ThS diluted in PBS and incubated for 2 h at RT in the dark. ZPs were then washed 3 x in PBS and mounted onto slides in 60 μl PBS drops and cover slipped. Congo Red was prepared as a 0.2% solution in 427 mM NaCl, filtered and stored in the dark. Isolated ZP were incubated overnight in 0.2% Congo Red in a watch glass at 4°C. ZP were then pelleted by centrifugation at 15000 x g for 5 min and the pellet resuspended in 0.25% SDS for 2 min to partially disperse the ZP. The ZP were centrifuged again and the pellet resuspended in PBS and allowed to dry on a slide. After washing with water to remove any remaining SDS, the ZP pellet was stained with 0.2% Congo Red for 2 hr at room temperature, washed several times with water, and then cover slips mounted using Fluoromount G. ThS and Congo Red stained ZP were examined with a Zeiss Axiovert 200 M microscope using filters with excitation at 425 nm and emission at >475 nm for ThS and excitation at 545–580, emission > 630 nm for Congo Red and images captured using a coupled device camera (CCD; AxioCam MRc, Zeiss) and Axiovision software version 4.5. Congo Red stained ZP were also imaged with differential interference contrast (DIC) to detect birefringence.
4
FFPE Tissue Immunofluorescence Labeling
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For IHC labelling of FFPE tissue sections, antigens were unmasked by heat-induced epitope retrieval (HIER) using either 10 mM citrate solution (pH 6.0) and/ or Tris ethylenediaminetetraacetic acid (TE) solution (pH 9.0). Tissue sections were blocked with 5% normal goat serum in phosphate-buffered saline (PBS; blocking buffer) before incubating with primary antibodies either overnight at 4 °C or for 2 h at RT (for primary antibody details, see Supp. Table S1 ). The resulting antigen–antibody complexes were detected using Alexa Fluor-conjugated secondary antibodies (1/400; AlexaFluor 488, 555, 647—secondary antibodies are listed in Supp. Table S1 ) and cell nuclei were labelled with DAPI (Invitrogen, UK). If applicable, tissue sections were then counterstained with 0.5% Thioflavin S (ThS; diluted in water; Sigma Aldrich, cat. no. T1892). Sections were mounted in fluorescence mounting media (Agilent, UK, cat. no. S302380-2) before imaging. All slides were imaged on the Akoya PhenoImager HT™ Automated Quantitative Pathology Imaging System (CLS143455) and images were processed using the InForm™ image analysis platform (Akoya Biosciences, US). Quantification analysis of the immunofluorescent signal was performed using HALO® (Indica Labs), a gold standard image analysis platform for quantitative analysis of IHC data. See Supplementary Materials and Methods for further details.
5
Amyloid Plaque Detection using Thioflavin S
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Thioflavin S (ThS; Sigma) is used to detect the deposition of amyloid plaque as described previously [16 (link)]. Briefly, dried sections were stained with fresh, filtered 1% ThS in water for 1 h, and then washed with 70% ethanol, water, and PBS. To co-stain with immunohistochemical staining, the sections were then incubated in blocking buffer and antibody dilution buffer with corresponding antibodies, as mentioned below.
6
Quantifying Amyloid-Beta Pathology in Mice
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In the same cryosections subjected to ex vivo and in vitro autoradiography, fibrillar Aβ deposits were measured with the histochemical dye, Thioflavin S (ThS; Sigma-Aldrich, St. Louis, MO, USA), as previously reported [9 (link)]. Aβ pathology was also evaluated in adjacent tissue sections by immunohistochemical staining with an anti-Aβ1-40 (1:400; Millipore Corp., Billerica, MA, USA) antibody, as previously reported [4 (link)]. The mice used for brain histology are presented in Table 2 . All sections were post-fixed in 4% paraformaldehyde for 30 min before staining. Fluorescent images were examined with a SteREO Lumar V.12 microscope (Carl Zeiss Microscopy GmbH, Jena, Germany), and the images were captured with a Zeiss Axiocam HRm S/N 1475 camera. Images of 3, 3′-diaminobenzidine stained sections were digitized with the Pannoramic 250 Flash II digital slide scanner (3DHistech, Budapest, Hungary), and the images were captured with the Pannoramic Viewer software.
7
Immunofluorescence Staining of Amyloid Plaques
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All mice were deeply anesthetized by intraperitoneal injection of 4% avertin for the brain extraction. After perfusion with 0.9% NaCl, mice were sacrificed by cervical dislocation and extracted brains were fixed in 4% paraformaldehyde (Biosesang, Korea). After 24 h of fixation, brains were immersed in 30% sucrose for 2 days. For immunofluorescence staining, 35 µm-thick frozen sections were incubated with 6E10 (Biolegend, USA, Catalog# SIG-39320, 1:200), 4G8 (Biolegend, USA, Catalog# SIG-39220, 1:200), AT8 (Invitrogen, USA, Catalog# MN1020, 1:200), or α-synuclein (BD Transduction Laboratories, USA, Catalog# 610786, 1:250) antibody diluted in 5% horse serum (Gibco, USA), then with Alexa555-conjugated secondary antibody (1:200 in PBS). Each stained section was incubated in 500 µM of Thioflavin S (ThS, Sigma-Aldrich, USA) dissolved in 50% ethanol for 7 min for ThS double-staining. Hoechst 33342 (10 µg/mL, Sigma-Aldrich, USA) was used to observe nuclear morphology. All images were taken using a fluorescence microscope (Leica DM2500, Germany). The number and area of plaques detected by ThS and 6E10 were quantified using Image J software.
8
Amyloid Plaque Quantification in Brain Tissue
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The hemisphere slices were stained with 0.5% fluorescent amyloid dye Thioflavin S (ThS, T1892, Sigma Aldrich) in the dark for 10 min followed by three washes with 50% ethyl alcohol and a final wash in PBS. Image analysis of the number and size of cortical and hippocampal ThS positive plaques was carried out on Image J FIJI software with “analyze particles” function.
9
Visualizing Protein Aggregation in E. coli
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E. coli BL21(DE3) cells harboring CarD or CarDtr expression vector and empty expression vector control were grown at 37 °C in LB medium until optical density (OD600) reached 0.5. The protein expression was induced by addition of 0.3 mM IPTG and the cultures were further grown at 37 °C for 5 h after induction. Thereafter, 0.5 mL of each bacterial culture was centrifuged at 6000 × g, 25 °C for 2 min. The bacterial cultures were washed thrice with 1 × PBS, followed by their fixation using 4% paraformaldehyde at 37 °C for 30 min. The fixed bacterial cultures were washed thrice with 1 × PBS and further incubated with 125 μM ThioflavinS (ThS, Catalog number T1892, Sigma) at 37 °C for 30 min in the dark. The bacterial cultures were again resuspended and washed thrice with 1 × PBS. 10 μL of the sample was placed on top of the glass slide, covered with a coverslip and air-dried. The images were acquired using confocal fluorescence microscope (Nikon A1R), using a 100 × oil-immersion objective and 1 Airy unit aperture. The ThS stained samples were excited using an excitation wavelength of 405 nm and an emission wavelength of 482 nm.
10
Amyloid-β Aggregation Characterization
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The sources of various reagents were as follows: Thioflavin-S (Th-S) (Sigma, USA), PKH26 Red Fluorescent Cell Linker Kit (Sigma Aldrich, USA), DiR (Beyotime, China), Amyloid-β42 peptides (GL Biochem, China, 95% purity), and FITC-labeled amyloid-β42 peptides (GL Biochem, China, 95% purity), Protease Inhibitor Cocktail Set III, EDTA-Free (Calbiochem, USA).
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