For IF analysis, mice were euthanized with CO2 and perfused with phosphate-buffered saline followed by 4 % paraformaldehyde solution. The brain was then removed, post-fixed in the same fixative overnight to 48 h and cut into 40 μm slices on a Leica VT1000S vibratome. IF on grafted brains was performed as described previously [31 (link)] using primary and secondary antibodies (Table S1 ). Antigen retrieval was performed by microwave boiling the slides in 10mM tri-Sodium Citrate buffer pH 6.0 (VWR). Aβ plaques were detected by staining with Thioflavin (Sigma). Briefly, for Thioflavin staining brain sections were incubated with a filtered 0.05 % aqueous Thioflavin-S (Sigma) solution in 50 % ethanol for 5 min at RT and rinsed gradually with 70 %, 95 % ethanol and water. Nuclei staining was performed using a specific anti-human Nuclear Antigen antibody (hNuclei) (Table S1 ), the pan-nuclear staining TOPRO3 (Invitrogen), or DAPI (Sigma). The sections were mounted with Glycergel (DAKO). Confocal images were obtained using a Nikon Ti-E inverted microscope equipped with an A1R confocal unit driven by NIS (4.30) software. The confocal was outfitted with 20 × (0.75 NA), 40x oil (1.4 NA) and 60x oil (1.4 NA) objectives lenses. For excitation 405 nm, 488 nm, 561 nm, 638 nm laser lines were used.
Thioflavin
Manufactured by Merck Group
Thioflavin is a fluorescent dye used in laboratory equipment. It is a small molecule that binds to specific protein structures, allowing for the detection and visualization of these structures in various experimental settings.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Hematoxylin and eosin, by Merck Group (1 mentions)
Amyloid precursor protein app, by Cell Signaling Technology (1 mentions)
Fluoro jade b, by Merck Group (1 mentions)
3 3 diaminobenzidine (dab), by Merck Group (1 mentions)
Mvx10, by Olympus (1 mentions)
Rabbit anti gfap z0334, by Agilent Technologies (1 mentions)
Mouse anti gfap mab360, by Merck Group (1 mentions)
Anti β actin 8226, by Abcam (1 mentions)
Milli q water, by Merck Group (1 mentions)
Truseq small rna sample preparation kit, by Illumina (1 mentions)
Protein g magnetic beads, by Thermo Fisher Scientific (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Wst 1, by Roche (1 mentions)
Homer1, by Abcam (1 mentions)
Pluronic f68, by Merck Group (1 mentions)
Glycergel, by Agilent Technologies (1 mentions)
Tri sodium citrate buffer ph 6, by Avantor (1 mentions)
Vt1000s vibratome, by Leica (1 mentions)
Ti e inverted microscope, by Nikon (1 mentions)
6 protocols using thioflavin
1
Immunofluorescence Imaging of Grafted Brains
2
Histological Analysis of Neurodegeneration
3
Western Blot and Immunohistochemistry of YKL-40 and Prion Proteins
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Detection of human YKL-40 by western blot was performed using the Human Chitinase 3-like 1 Antibody AF2599 (R&D Biosystems). Detection of murine YKL-40 by western blot, and human and murine YKL.40 by immunohistochemistry and immunofluorescence was performed using the Chitinase 3-like 1 antibody PA5–43746 (Thermo Fisher).
Anti-GAPDH 9484, anti-β-actin 8226 and anti-vimentin 92547 antibodies were from Abcam. Mouse anti-GFAP MAB360 was from Millipore. Rabbit Anti-GFAP Z0334 and anti-amyloid beta (β-amyloid) M0872 were from Dako. Anti-PrP(12F10) A03221 was from SPI Bio. Thioflavin was from Sigma. Chimeric recombinant prion protein (PrP), composed of the Syrian hamster residues 23–137 followed by sheep residues 141–234 (of the R154 Q171 polymorphism), was from Thermo Fisher.
Anti-GAPDH 9484, anti-β-actin 8226 and anti-vimentin 92547 antibodies were from Abcam. Mouse anti-GFAP MAB360 was from Millipore. Rabbit Anti-GFAP Z0334 and anti-amyloid beta (β-amyloid) M0872 were from Dako. Anti-PrP(12F10) A03221 was from SPI Bio. Thioflavin was from Sigma. Chimeric recombinant prion protein (PrP), composed of the Syrian hamster residues 23–137 followed by sheep residues 141–234 (of the R154 Q171 polymorphism), was from Thermo Fisher.
4
Amyloid Fibril Formation Assay
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BI was purchased from Sigma Chemical Co.
(catalog number I6634). Milli-Q water (Millipore Ltd., Bedford, MA)
was used in all experiments to prepare the samples. Thioflavin and
tyrosine were obtained from Sigma-Aldrich. The stock solution of BI
(2 mg/mL) was prepared freshly in NaCl/HCl solution of pH ∼
1.6 (25 mM HCl containing 0.1 M NaCl). A stock solution of ThT was
prepared, and concentration was checked using a molar extinction coefficient
of 24,420 M–1 cm–1 at 420 nm.
ThT was stored at 4 °C and protected from light for further use.
Aliquots of the protein samples at specified intervals were collected
and used for the spectroscopic analyses.
(catalog number I6634). Milli-Q water (Millipore Ltd., Bedford, MA)
was used in all experiments to prepare the samples. Thioflavin and
tyrosine were obtained from Sigma-Aldrich. The stock solution of BI
(2 mg/mL) was prepared freshly in NaCl/HCl solution of pH ∼
1.6 (25 mM HCl containing 0.1 M NaCl). A stock solution of ThT was
prepared, and concentration was checked using a molar extinction coefficient
of 24,420 M–1 cm–1 at 420 nm.
ThT was stored at 4 °C and protected from light for further use.
Aliquots of the protein samples at specified intervals were collected
and used for the spectroscopic analyses.
5
Molecular Toolkit for Cell Studies
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List of Taqman probes assays, Exiqon primer sets and antibodies is given in S7 Table . Lipofectamine 2000 was from Thermo Fisher Scientific. Thioflavin and Propidium Iodide were from Sigma. WST-1 was from Roche. TruSeq Small RNA Sample Preparation Kit was from Illumina. CHROMA SPIN-200 spin columns were from Clontech and Protein G magnetic beads were from Invitrogen.
6
Peptide-Loaded PLGA Nanoparticles
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Poly(d ,l -lactide-co-glycolide) acid (PLGA RG-503H 50:50, inherent viscosity in 0.1% (w/v) CHCl3 at 25 °C = 0.38 dL/g) was used as received from the manufacturer (Boehringer-Ingelheim, Ingelheim am Rhein, Germany). According to the experimental titration results of the carboxylic end of the polymers (4.94 mg KOH/g polymer), the molecular weight of RG-503H was calculated to be 11,000 Da. Peptide KLVFF (Lys-Leu-Val-Phe-Phe, C42H63N9O8, MW 822.01) was obtained from Mimotopes (Springvale Rd Mulgrave, Victoria, Australia). Pluronic F68 (molecular weight of 8500–9000 Da) was purchased from Sigma-Aldrich. Primary antibodies were purchased from Synaptic Systems (Homer1) and Abcam (MAP2). Fetal bovine serum (FBS) and phosphate-buffered saline (PBS) were purchased from Euroclone Celbio (Milan, Italy). Secondary Alexa Fluor conjugated antibodies were purchased from Invitrogen. Thioflavin was obtained from Sigma Aldrich. Synthetic Aβ peptide, human Aβ(1–42), was purchased from Abcam. All the solvents were of analytical grade, and all other chemicals and media were used as received from the manufacturers, and unless otherwise indicated, obtained from Sigma-Aldrich.
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