Sections were pre-treated in 70% formic acid and incubated with anti-Iba1 (Wako, Osaka, Japan) (1:1000) and anti-Aβ (4G8, Covance, Greenfield, IN, USA) (1:2000) antibodies overnight at 4 °C in 0.5% BSA, followed by incubation with Alexa Fluor 488 and Alexa Fluor 594 (Molecular Probes, OR, USA) (1:1000). Sections were photographed using a Laser Olympus U-RFL-T fluorescent microscope (Olympus, Japan) and MMIcellTools software. Microglia burden in SP-free areas as well as in the close proximity of SP (up to 50 μm) was quantified as previously described [12 (link)].
Anti aβ 4g8
Manufactured by Fortrea
Sourced in Japan
The Anti-Aβ (4G8) is a lab equipment product that recognizes and binds to the amyloid-beta (Aβ) peptide. It is a widely used tool in the study of Alzheimer's disease and other neurodegenerative conditions.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488, by Thermo Fisher Scientific (3 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (3 mentions)
Anti iba1, by Fujifilm (3 mentions)
Laser u rfl t fluorescent microscope, by Olympus (2 mentions)
Alexa488 conjugated anti rabbit igg, by Jackson ImmunoResearch (2 mentions)
Anti α actin, by Abcam (2 mentions)
Alexa 647 conjugated anti rabbit igg, by Jackson ImmunoResearch (2 mentions)
Lysotracker red, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 555 conjugated goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Cellmask deep red plasma membrane stain, by Thermo Fisher Scientific (1 mentions)
6 protocols using anti aβ 4g8
1
Immunohistochemical Analysis of Microglia Burden
2
Immunohistochemical Analysis of Microglial Activation and Amyloid-Beta Pathology
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Right hemisphere sections were selected at 1.5, 0.5, − 0.5, − 1.5, − 2.5, and − 3.5 mm from Bregma [32 ]. Sections were pretreated with formic acid (70%) and incubated with anti-Iba1 (Wako, Osaka, Japan) (1:1000) and anti-Aβ (4G8, Covance, Greenfield, IN, USA) (1:2000) antibodies at 4 °C in 0.5% BSA overnight. Alexa Fluor 594 and Alexa Fluor 488 (Molecular Probes, OR, USA) (1:1000) were used as secondary antibodies. A Laser Olympus U-RFL-T fluorescent microscope (Olympus, Japan) and MMIcellTools v.4.3 (Molecular Machines and Industries, Eching, Germany) software was used was used to for image acquisition. Senile plaque (SP) burden and microglia burden in proximity of (within 50 μm) and far from (> 50 μm) SP were measured using Image J software as previously described [17 (link)].
3
Congo Red Fluorescence Imaging of Amyloid Deposits
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For Congo Red fluorescence imaging, the brains were fixed and washed as above then stained with 0.2 mg/ml Congo Red in PBS for 10 min. The Congo Red solution was freshly prepared and filtered through a 0.2 μm filter before use. After PBS washing, brains were observed using confocal microscopy. Congo Red fluorescence was detected as described (Wiesehan et al., 2003 (link)). LysoTracker red (Molecular Probes) staining was performed as previously described (Ling et al., 2009 (link)). For the CellMask plasma membrane staining, whole brains were first immunostained using anti-Aβ 4G8 (Covance) and Alexa Fluor-555-conjugated goat anti-mouse IgG (Invitrogen) secondary antibody, then washed in PBS, followed by a 10 min incubation in PBS containing 5 μg/ml CellMask™ Deep Red plasma membrane stain (Invitrogen). The brains were not permeabilized with detergent prior to the CellMask staining. After 5×PBS washing, brains were mounted and observed by confocal microscopy.
4
Quantifying Amyloid-β Burden and Smooth Muscle Cell Fragmentation in Pial Vessels
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To quantify CAA burden and Aβ-associated fragmentation of smooth muscle cells in pial vessels [20 (link), 31 (link)], tangential brain sections were incubated with anti-Aβ (4G8, 1:1,000, mouse; Covance) and the smooth muscle marker anti-α-actin (1:300, rabbit, Abcam) for 48 h. After washing, sections were labeled with Alexa 488-conjugated anti-rabbit IgG (1:200; Jackson ImmunoResearch) and Alexa 647-conjugated anti-rabbit IgG (1:200; Jackson ImmunoResearch). Pial arterioles (n = 30–50/group) positive for Aβ and α-actin, ranging in diameter from 20 to 100 μm, were randomly imaged by confocal microscope (63x). Aβ accumulation around pial-penetrating arteries was assessed by the ratio (%) between 4G8+ Aβ immunoreactivity and α-actin+ pial vessel area obtained with ImageJ in the same sections in which smooth muscle fragmentation was quantified. However, the A β specie (1–40, 1–42) was not determined. Smooth muscle cell fragmentation was quantified by counting α-actin fragments in pial-penetrating arteriole using ImageJ, expressed as the fragmentation index: 100 − [(1/number of α-actin fragments) × 100].
5
Quantifying Microglial Response to Amyloid Plaques
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After pretreatment in 70% formic acid, sections were incubated in anti-Iba1 (Wako, Osaka, Japan) (1:1.000) and anti Aβ (4G8, Covance, Greenfield, IN, USA) (1:2000) antibodies overnight at 4º C in 0.5% NGS. Secondary antibodies Alexa Fluor 594 and Alexa Fluor 488 (Molecular Probes, OR, USA) (1:1.000) were used. Sections were mounted and photographed using a fluorescent microscope (Laser Microdissection System CellCut Version 4.3, Molecular Machines & Industries AG, Switzerland). SP burden was analyzed in the cortex and hippocampus. Microglia burden was also quantified using Image J software. Regions of interest were marked around SP (up to 50µm from the plaque border) to quantify microglia burden in the proximity of amyloid deposits. Microglia burden was also measured in random SP-free areas from APP/PS1 and APP/PS1xdb/db mice, and in animals without plaques (Control and db/db mice), as previously described [14, 24] (Control n=5, Control-PP n=5, APP/PS n=4, APP/PS1-PP n=4, db/db n=5, db/db-PP=4, APP/PSxdb/db n=4, APP/PS1xdb/db-PP n=5). .
6
Quantification of Aβ-Induced Smooth Muscle Fragmentation
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To quantify Aβ-associated fragmentation of smooth muscle cells in pial vessels [20 (link), 30 (link)], brain sections were incubated with anti-Aβ (4G8, 1:1,000, mouse; Covance) and the smooth muscle marker anti-α-actin (1:300, rabbit, Abcam) for 48 hr. After washing, sections were labeled with Alexa 488-conjugated anti-rabbit IgG (1:200; Jackson ImmunoResearch) and Alexa 647-conjugated anti-rabbit IgG (1:200; Jackson ImmunoResearch). Pial arterioles (n = 30–50/group) positive for Aβ and α-actin, ranging in diameter from 20 to 100 µm, were randomly imaged by confocal microscope (63x). The fragmentation of smooth muscles was quantified by counting the number of α-actin fragments of each arteriole using ImageJ, expressed as the fragmentation index: 100 − [(1/number of α-actin fragments) x 100].
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