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Methoxy x04

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Methoxy-X04 is a fluorescent dye used for the detection of amyloid-beta deposits in biological samples. It functions by selectively binding to amyloid-beta fibrils, allowing for their visualization and quantification.

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Lab products found in correlation

Anti rabbit igg dylight 549, by Vector Laboratories (3 mentions) Anti iba1, by Fujifilm (2 mentions) Donkey anti rabbit fitc, by Jackson ImmunoResearch (2 mentions) Cy3 conjugated streptavidin, by Thermo Fisher Scientific (2 mentions) A1rsi confocal microscope, by Nikon (2 mentions) Percoll, by Merck Group (2 mentions) Texas red dextran, by Thermo Fisher Scientific (2 mentions) Anti β amyloid 1 16, by BioLegend (1 mentions) Fluoromount g, by Southern Biotech (1 mentions) A1r confocal microscope, by Nikon (1 mentions) Cytoflex lx, by Beckman Coulter (1 mentions) Legend max β amyloid x 42 elisa kit, by BioLegend (1 mentions) 4 hydroxytamoxifen 4 oht, by Merck Group (1 mentions) Alexa fluor 647 anti rabbit igg, by Thermo Fisher Scientific (1 mentions) Anti spp1, by R&D Systems (1 mentions) Streptavidin alexa fluor 647, by Thermo Fisher Scientific (1 mentions) Matlab, by MathWorks (1 mentions) Opal 570, by Akoya Biosciences (1 mentions) Opal 520, by Akoya Biosciences (1 mentions) Prolong gold antifade mountant, by Thermo Fisher Scientific (1 mentions)

27 protocols using methoxy x04

1

Immunohistochemical Analysis of Microglia and Amyloid Plaques in Mouse Brains

Cited 2 times
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Brain samples were processed as previously described [3 (link)]. Briefly, mice were anesthetized with isoflurane and perfused with ice-cold PBS containing 1 U/ml of heparin. Brains were fixed in 4% PFA for 48 h at 4 °C, rinsed with PBS, and incubated for 48 h at 4 °C in 30% sucrose before freezing in a 2:1 mixture of 30% sucrose and optimal cutting temperature compound. Brain samples were sectioned (40 μm) on a cryostat and stained with anti-Iba1 (Wako, Cat. No. 019-19741) and incubated overnight at 4 °C. Sections were washed with PBS and incubated with anti-rabbit IgG DyLight 549 (Vector Laboratories, Cat. No. DI-1549), methoxy-X04 (Tocris Bioscience, Cat. No. 4920) and TO-PRO-3 (Thermo Fisher Scientific, Cat. No. T3605) for 1 h at room temperature. The methoxy-X04 plaque staining was confirmed by staining with purified anti-β-Amyloid 1–16 (BioLegend, Cat. No. 803001, clone 6E10). Sections were washed and mounted using Fluoromount G (SouthernBiotech, Cat. No. 0100-001). Random images were taken of the cortices immediately dorsal to the hippocampus as previously described [3 (link), 56 (link)]. Images were taken using a Nikon A1R confocal microscope at the University of Wisconsin Optical Imaging Core. Images were then processed with Imaris 9.2.1 (Bitplane).
Shippy D.C., Wilhelm C., Viharkumar P.A., Raife T.J, & Ulland T.K. (2020). β-Hydroxybutyrate inhibits inflammasome activation to attenuate Alzheimer’s disease pathology. Journal of Neuroinflammation, 17, 280.
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2

In vivo Aβ Phagocytosis Assay

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In vivo Aβ phagocytosis assay was performed as previously described (25 (link), 26 (link)). Briefly, 5XFAD and OPN-KO.5XFAD mice were injected intraperitoneally with methoxy-X04 (Tocris Bioscience) at 10 mg/kg in 50% DMSO/50% NaCl (0.9%), pH 12. Brains were isolated 3 h after injection and made into single-cell suspensions followed by analysis of frequencies of methoxy-X04+ microglia by flow cytometry using a CytoFLEX LX (Beckman Coulter) followed by analysis using FlowJo (Tree Star).
Qiu Y., Shen X., Ravid O., Atrakchi D., Rand D., Wight A.E., Kim H.J., Liraz-Zaltsman S., Cooper I., Schnaider Beeri M, & Cantor H. (2023). Definition of the contribution of an Osteopontin-producing CD11c+ microglial subset to Alzheimer’s disease. Proceedings of the National Academy of Sciences of the United States of America, 120(6), e2218915120.
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3

Quantifying Soluble and Insoluble Amyloid-beta

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The concentrations of soluble and insoluble β-Amyloid were measured using the LEGEND MAX™ β-Amyloid x-42 ELISA kit (Biolegend) according to the manufacturer’s instructions. Specifically, brain samples were homogenized using a glass homogenizer with 6 passes on ice in TBS containing protease inhibitors. Samples were centrifuged at 350000xg for 20min. The supernatant containing soluble β-Amyloid was collected. The pellet was resuspended in TBS containing 1% triton and centrifuged again for collection of insoluble β-Amyloid. ELISA assays were performed immediately.
For measurement of in vivo β-Amyloid uptake, mice were injected i.p. with methoxy-X04 (4920, Tocris Bioscience) at the dose of 10mg/kg. Mice were euthanized at 3h after methoxy-X04 injection and flow cytometry analysis were performed to examine microglia uptake of methoxy-X04.
Zhang Y., Fung I.T., Sankar P., Chen X., Robison L.S., Ye L., D’Souza S.S., Salinero A.E., Kuentzel M.L., Chittur S.V., Zhang W., Zuloaga K.L, & Yang Q. (2020). Depletion of NK cells improves cognitive function in the Alzheimer’s Disease mouse model. Journal of immunology (Baltimore, Md. : 1950), 205(2), 502-510.
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4

Inducing Recombination and Plaque Labeling in Mice

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Recombination was induced with 4-hydroxytamoxifen (4-OHT) (Sigma, St. Louis, MO). 4-OHT was dissolved by sonication in 10% EtOH/90% corn oil at a concentration of 10 mg/ml; each mouse was injected intraperitoneally (i.p.) with 200 μl (2 mg) (Cazzulino et al., 2016 (link)).
Mice were given an i.p. injection of Methoxy-X04 (5 mg/kg; Tocris Bioscience, Minneapolis, MN) 24 h prior to sacrifice to label plaques (Meyer- Luehmann et al., 2008 (link)). This procedure was adapted from Rudinskiy et al. (2012) (link).
Perusini J.N., Cajigas S.A., Cohensedgh O., Lim S.C., Pavlova I.P., Donaldson Z.R, & Denny C.A. (2017). Optogenetic stimulation of dentate gyrus engrams restores memory in Alzheimer's disease mice. Hippocampus, 27(10), 1110-1122.
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5

Methoxy-X04 Modulates Amyloid-Beta Dynamics

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Methoxy-X04 (Tocris Bioscience, Bristol, UK) was administered intraperitoneally (IP; 10 mg/kg) and after 60 minutes Aβ40 was intracisternally injected. The brain was analyzed after an addition 30 minutes.
Peng W., Achariyar T.M., Li B., Liao Y., Mestre H., Hitomi E., Regan S., Kasper T., Peng S., Ding F., Benveniste H., Nedergaard M, & Deane R. (2016). Suppression of glymphatic fluid transport in a mouse model of Alzheimer’s disease. Neurobiology of disease, 93, 215-225.
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6

Generation and Characterization of Syk and Trem2 Mutant Murine Models

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For generation of Sykfl/fl- and SykiΔMG-5×FAD mice, 1- or 4-month-old littermates were fed a tamoxifen (TAM) diet for 4 weeks followed by replacement with regular chow. Treated mice were sacrificed at 6-, 9- or 5-, 9-months of age as indicated in the RESULTS. To determine fibrillar Aβ phagocytosis by microglia in vivo, 6-month-old Sykfl/fl- and SykiΔMG-5×FAD mice were intraperitoneally injected with methoxy-X04 (Tocris) at 10 mg/kg in a PBS/DMSO mixture. Mice were sacrificed after 3 hours of injection and brains were made into single-cell suspension. For the short term of anti-CLEC7A antibody treatment, 8-month-old Trem2R47H-5×FAD mice received intraperitoneal injection of anti-CLEC7A antibody (2A11), or anti-human ILT1 antibody (Fc mutated, clone 135.5) (Molgora et al., 2020 (link)) as a control, for 4 times within 10 days. Mice were sacrificed within 24 hours after the last injection for assessment. For testing the in vivo effect of 2A11 treatment on fibrillar Aβ phagocytosis by microglia Trem2R47H-5×FAD mice were first injected ip. with a single dose of 2A11 followed by ip. injection of methoxy-X04 after 24 hours.
Wang S., Sudan R., Peng V., Zhou Y., Du S., Yuede C.M., Lei T., Hou J., Cai Z., Cella M., Nguyen K., Poliani P.L., Beatty W.L., Chen Y., Cao S., Lin K., Rodrigues C., Ellebedy A.H., Gilfillan S., Brown G.D., Holtzman D.M., Brioschi S, & Colonna M. (2022). TREM2 Drives Microglia Response to Amyloid-β via SYK-dependent and -independent Paths. Cell, 185(22), 4153-4169.e19.
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7

Quantification of Microglia Phagocytosis

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Mice were intraperitoneally administered methoxy-X04 (4920; Tocris Bioscience, Bristol, UK) at a concentration of 10 mg/kg and dissolved in a 10 % DMSO and 90 % PBS solution (pH 12) for 3 h. Brain single-cell suspensions were prepared as outlined in the “Isolation of mouse adult microglia and astrocytes from brains” section. Cells were then incubated with CD16/CD32 Fc receptor-blocking antibody for 15 min at 4 °C to minimize nonspecific binding. Post-blocking, cells were stained with CD11b-APC (1:100) and CD45-FITC (1:100) in 200 μL PBS for 1 h at 4 °C. After a final wash, they were resuspended in 2 % FBS/PBS and subjected to flow cytometry on a CytoFlex S, with data analysis performed via FlowJo. methoxy-X04-injected WT mice were used as controls to set the threshold for identifying non-phagocytosing cells.
Ouyang P., Cai Z., Peng J., Lin S., Chen X., Chen C., Feng Z., Wang L., Song G, & Zhang Z. (2024). SELENOK-dependent CD36 palmitoylation regulates microglial functions and Aβ phagocytosis. Redox Biology, 70, 103064.
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8

Dendritic Spine Turnover Quantification

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Dendritic spine turnover was quantified as described previously (Bittner et al., 2012 (link)), with modifications. 5XFAD and hAPP-J20 mice were stereotactically injected with AAV1.hSyn.eGFP.WPRE.bGH in the somatosensory cortex to fluorescently label dendrites and spines. Following the injection, a craniotomy was prepared as described above (“Cranial window surgery”). Alexa 594-labeled fibrinogen (1.5 mg/mL; ThermoFisher Scientific) was administered i.v. at day 12 and day 13 following surgery; Methoxy-X04 (5 mg/kg; Tocris Biosciences) was administered i.p. on day 13 following surgery to label Aβ plaques. Dendritic spines with and without Alexa 594-labeled fibrinogen deposits and which were located <50 and >50 μm of an Aβ plaque were imaged using 2P microscopy at 840 nm and 920 nm and a 40 × objective at 4 × optical zoom as described under “In vivo brain imaging and cortical microinjections”. The same location was imaged 14 days later.
Merlini M., Rafalski V.A., Coronado P.R., Gill T.M., Ellisman M., Muthukumar G., Subramanian K.S., Ryu J.K., Syme C.A., Davalos D., Seeley W.W., Mucke L., Nelson R.B, & Akassoglou K. (2019). Fibrinogen Induces Microglia-Mediated Spine Elimination and Cognitive Impairment in an Alzheimer’s Disease Model. Neuron, 101(6), 1099-1108.e6.
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9

Visualizing Biotinylated Antibody Binding

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Mice were injected i.p. with 800 μg/mouse of biotinylated 5B8 every 2 days (3 total doses). Mice were perfused with saline, and spinal cord or brain were processed for fresh frozen sections as described. Sections were fixed with 4% PFA for 10 min and biotinylated 5B8 was detected using Cy3-conjugated streptavidin (1:100; Invitrogen) for 30 min at 25 ˚C. Sections were incubated for 1h with fibrinogen antibody (1:2000), followed by FITC donkey anti-rabbit (1:500; Jackson ImmunoResearch) for 30 min at 25 ˚C. For amyloid plaque staining, sections were counterstained with Methoxy-X04 (Tocris; 4% vol of 10 mg/ml).
Ryu J.K., Rafalski V.A., Meyer-Franke A., Adams R.A., Poda S.B., Coronado P.E., Pedersen L.Ø., Menon V., Baeten K.M., Sikorski S.L., Bedard C., Hanspers K., Bardehle S., Mendiola A.S., Davalos D., Machado M.R., Chan J.P., Plastira I., Petersen M.A., Pfaff S.J., Ang K.K., Hallenbeck K.K., Syme C., Hakozaki H., Ellisman M.H., Swanson R.A., Zamvil S.S., Arkin M.R., Zorn S.H., Pico A.R., Mucke L., Freedman S.B., Stavenhagen J.B., Nelson R.B, & Akassoglou K. (2018). Fibrin-targeting immunotherapy protects against neuroinflammation and neurodegeneration. Nature immunology, 19(11), 1212-1223.
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10

Multimodal Imaging of Microglial Responses

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Floating sections were blocked with 3% BSA and 0.25% Triton X-100 in PBS, and then stained with anti–Iba-1 (rabbit polyclonal, 1:5,000; Wako; or goat polyclonal, 1:1,000; Abcam), anti–human TREM2 ECD (goat polyclonal, 1:500; R&D Systems), anti–human TREM2 C terminus (D814C rabbit mAb, 1:500; Cell Signaling), anti-Spp1 (goat polyclonal, 1:500; R&D Systems), anti-APP (22C11 mouse mAb, 1:1,000; Millipore) and/or anti-NeuN (D3S3I rabbit mAb, 1:500; Cell Signaling) overnight at 4°C followed by staining with anti–rabbit IgG DyLight 549 (1:2,000; Vector), anti–goat IgG Alexa Fluor 488 (1:2,000; Abcam), anti–rabbit IgG Alexa Fluor 647 (goat polyclonal, 1:1,000; Invitrogen), anti–goat IgG-biotin (donkey polyclonal, 1:2,000; Invitrogen), streptavidin Alexa Fluor 647 (1:2,000; Invitrogen), methoxy-X04 (3 µg/ml; Tocris), and/or TO-PRO-3 iodide (300 nM; Thermo Fisher Scientific) for 1 h at room temperature. All antibodies were used in blocking buffer, and between all incubations, sections were washed for 10 min in PBS three times. Images were collected using a Nikon A1Rsi+ confocal microscope. Three-dimensional image segmentation of microglia, plaques, and neurons, and extraction of parameters were performed in Imaris 8.1 (Bitplane), and further processing was performed using automated scripts in Matlab (Mathworks). For detailed image analysis procedures, see Supplemental Methods.
Song W.M., Joshita S., Zhou Y., Ulland T.K., Gilfillan S, & Colonna M. (2018). Humanized TREM2 mice reveal microglia-intrinsic and -extrinsic effects of R47H polymorphism. The Journal of Experimental Medicine, 215(3), 745-760.
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