Fixed tissue was sectioned on a vibratome (Leica Biosystems, Buffalo Grove, IL) at 50 μm. Sequential sections were collected and stored in PBS with 0.02% NaN3 until used. CAA was visualized by immunohistochemistry for Aβ1–40 (Invitrogen, Camarillo, CA, 1:5000) as described previously[31 (link)]. Briefly, free-floating sections were pretreated with 90% formic acid for 4 min and then incubated overnight with the primary antibody, incubated with anti-rabbit secondary antibody (Vector Laboratories, Burlingame, CA). The signal was amplified and visualized with an avidin-biotin complex peroxidase kit (Vector Laboratories, Burlingame, CA), and 3,3’ diaminobenzidine substrate kit (Vector Laboratories, Burlingame, CA). Sections were mounted on glass slides and coverslipped with Depex mounting media.
Aβ1 40
Manufactured by Thermo Fisher Scientific
Sourced in United States
Aβ1–40 is a synthetic peptide that corresponds to the first 40 amino acids of the amyloid-beta (Aβ) protein. It is commonly used in research related to Alzheimer's disease and other neurodegenerative disorders.
Automatically generated - may contain errors
Lab products found in correlation
Aβ1 42, by Thermo Fisher Scientific (6 mentions)
Aβ1 42 elisa kit, by Thermo Fisher Scientific (3 mentions)
Anti rabbit secondary antibody, by Vector Laboratories (1 mentions)
Vibratome, by Leica (1 mentions)
3 3 diaminobenzidine substrate kit, by Vector Laboratories (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Interleukin 6 (il 6), by Abcam (1 mentions)
Trem2, by Abcam (1 mentions)
Brain derived neurotrophic factor (bdnf), by Abcam (1 mentions)
Ptau ser396, by Thermo Fisher Scientific (1 mentions)
Tnf α, by Abcam (1 mentions)
Tdp 43, by Proteintech (1 mentions)
Neurofilament light, by UmanDiagnostics (1 mentions)
Anti actin, by Transgene (1 mentions)
Anti actin, by Merck Group (1 mentions)
Anti vegfr2, by Cell Signaling Technology (1 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
17 protocols using aβ1 40
1
Immunohistochemical Visualization of CAA
2
Quantifying Amyloid-Beta Levels in APP/PS1 Mice
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Aβ1-40 and Aβ1-42 levels in brains, serum, and cerebrospinal fluid (CSF) of APP/PS1 mice were measured with Aβ1-40 and Aβ1-42 ELISA kit (KHB3481 and KHB3441, Invitrogen, United States), respectively. Protein concentration was measured by Pierce™ BCA Protein Assay Kit (Catalog: 23225, Thermo Fisher Scientific). Briefly, the brain tissue was homogenized in PBS (0.01 M) with 1 mM EDTA, 1% BSA (Sigma, United States), 2% Triton X-100 and protease inhibitor cocktail (Sigma) at 14000× g for 30 min at 4°C. The supernatant was collected for the assay of soluble proteins. The insoluble pellet was dissolved in guanidine hydrochloride and centrifuged at 1,00,000×g for 1 h at 4°C, and the resulting supernatant was collected for the assay of insoluble protein. The ELISA assay has been extensively tested, and no cross-reactivity between Aβ1-40 and Aβ1-42 was found.
3
Microglia Isolation and Activation
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Microglia were prepared as previously described [21 (link)]. Briefly, mixed glia were isolated from triturated and filtered cortical tissue of C57BL/6 neonatal mice. Tissue was centrifuged (800 × g, 5 min), the pellet was resuspended in pre-warmed media, triturated and cells were seeded (1 ml) and cultured in T25 cm2 flasks in cDMEM (31,330–038, Bioscience, USA) supplemented with macrophage colony stimulating factor (M-CSF; 100 ng/ml; 416-ML, R&D Systems, UK), and granulocyte macrophage colony stimulating factor (GM-CSF; 100 ng/ml; 415-ML, R&D Systems, UK) for 10–12 days with media change every 3–4 days. Cells were shaken for 2–3 h (37 °C) to dislodge non-adherent microglia and, after centrifuging (800 × g, 5 min), the pellet was harvested, cells counted, seeded in 24-well plates (1 × 105 cells/well), and cultured for a further 2 days.
Cells were incubated for 24 h in the presence or absence of IFNγ (12.5 ng/ml) + and amyloid-β (Aβ; 10 μM comprising 4.8 μM Aβ1–40 and 5.8 μM Aβ1–42; Invitrogen, USA). Aβ was prepared as follows: lyophilised Aβ1–40 and Aβ1–42 peptides were dissolved in HPLC grade water to give a stock solution (6 mg/ml), which was diluted using sterile PBS (final concentration 1 mg/ml). This was allowed to aggregate (24 h, 220 rpm, 37 °C) to give a mix that was shown, by enhanced Thioflavin T binding, to contain both oligomers and fibrils (Supplementary Fig.1 ).
Cells were incubated for 24 h in the presence or absence of IFNγ (12.5 ng/ml) + and amyloid-β (Aβ; 10 μM comprising 4.8 μM Aβ1–40 and 5.8 μM Aβ1–42; Invitrogen, USA). Aβ was prepared as follows: lyophilised Aβ1–40 and Aβ1–42 peptides were dissolved in HPLC grade water to give a stock solution (6 mg/ml), which was diluted using sterile PBS (final concentration 1 mg/ml). This was allowed to aggregate (24 h, 220 rpm, 37 °C) to give a mix that was shown, by enhanced Thioflavin T binding, to contain both oligomers and fibrils (Supplementary Fig.
4
Measurement of Aβ Peptides in APP-SY5Y Cells
5
Biomarker Validation in Rhesus Monkeys
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Validation of human ELISAs using rhesus monkey samples were validated in our previous study7 . Aβ1‐42, Aβ1‐40, t‐tau, p‐tau Ser199, p‐tau Thr231, p‐tau Ser396 (Invitrogen), TNF‐α, IL‐6, TREM2, BDNF (Abcam), TDP‐43 (Proteintech), and neurofilament‐light (Uman Diagnostics) ELISA assays were performed according to each kit manufacturer's instructions, after sample dilution optimization.
6
Amyloid-beta Peptide Preparation
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The following primary antibodies were used: anti-Notch1 intracellular domain (NICD) (Cell Signaling Technology, MA, USA), anti-Notch1 extracellular domain (NEXT) (Thermo Fisher Scientific, MA, USA), anti-Hey-1 (GeneTex, CA, USA), anti-Actin (TransGen, Beijing, China). Synthetic amyloid-beta peptides 1–40 (Aβ1–40) were purchased from Invitrogen (Invitrogen, CA, USA) and dissolved in hexafluoreisopropanol (HFIP) (Sigma, MO, USA) for 2 h at room temperature, and lyophilized peptide was dissolved in dimethylsulfoxide (DMSO).
7
Antibody Characterization for VEGFR
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The following primary antibodies were used: anti-VEGFR2 (Cell Signaling Technology, MA, USA, 9698S), anti-VEGFR1 (GeneTex, CA, USA, GTX61100), anti-Actin (Millipore Corporation, MA, USA, MAB1501). Specific antibody to splicing variant form of VEGFR2 (sVEGFR2) was purchased from Acris Antibodies (Herford, Germany, AP26034PU-L). Amyloid-beta peptides 1–40 (Aβ1–40) were purchased from Invitrogen (CA, USA) and dissolved in hexafluoreisopropanol (HFIP) for 2 h at room temperature, and lyophilized peptide was dissolved in dimethylsulfoxide (DMSO).
8
Quantifying Brain Amyloid-beta Levels
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Aβ concentration in total brain was measured by an Aβ1–40 and an Aβ1–42 enzyme-linked immunosorbent assay (ELISA) kits (both Invitrogen, Camarillo, CA, USA). Sample preparation, processing, and detection were performed according to the manufacturer’s instructions. Statistical analysis used a one-way ANOVA with one variable (treatment) followed by a Tukey multiple comparison post hoc test. Statistical significance was set at P ≤ 0.05. All results are expressed as means ± SEM.
9
Cerebral Cortex Aβ Measurement
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The cerebral cortex was isolated from mice and subjected to Aβ measurement with the use of Aβ1‐40 or Aβ1‐42 enzyme‐linked immunosorbent assay kit according to the manufacturer's instructions (Invitrogen, USA). The soluble Aβ fraction probably contained monomeric and oligomeric Aβ. Insoluble Aβ was treated with 5 M guanidine/50 mM Tris‐HCL (pH 8.0) buffer before ELISA measurement (Martorell et al., 2019 ).
10
Amyloid-beta Quantification in Mouse Brain
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