71-5800

antibody from Invitrogen Antibodies

Targeting: APP AD1

Provider product page for 71-5800

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Immunoprecipitation

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Antibody data

Antibody Data
Antigen structure
References [59]
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Validations
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Product number: 71-5800 - Provider product page
Provider: Invitrogen Antibodies
Product name: beta Amyloid Polyclonal Antibody
Antibody type: Polyclonal
Antigen: Synthetic peptide
Reactivity: Human
Host: Rabbit
Isotype: IgG
Vial size: 100 µg
Concentration: 0.25 mg/mL
Storage: -20°C

APP protein structure - 71-5800 shown in red.

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Experimental details: Figure 10 Analysis of the plaque load shows an age-dependent increase of plaque numbers in the hippocampus of APPPS1 mice. A one-way ANOVA showed an overall difference of p

Supportive validation

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Experimental details: Figure 10 Histological analysis of biomarkers in the brain . Top half of figure: effect of D-Ala2 glucose-dependent insulinotropic polypeptide (GIP) treatment on three hallmarks of Alzheimer's disease in 6-month-old APP/PS1 mice. ( A ) Quantification of beta-amyloid plaque, ( B ) of dense core plaque load in the cortex of APP/PS1 and, ( C ) of activated microglia (neuroinflammation) in the cortex of APP/PS1 mice. The percentage of stained cortex area was used as measurement for these three hallmarks. Data show mean +- standard error of the mean (SEM) of six mice per group (Student's unpaired t- test, * P < 0.05). Micrographs: The figures on the top row illustrate examples of the cortex of APP/PS1 mice injected with ( D ) saline and ( E ) D-Ala 2 GIP, immunohistologically stained against beta-amyloid (scale bar: 880 mum). The figures in the middle row illustrate example of cortex of APP/PS1 mice injected with ( F ) saline and ( G ) D-Ala 2 GIP, stained with Congo red (scale bar: 880 mum). The figures on the bottom row illustrate examples of cortex of APP/PS1 mice injected with ( H ) saline and ( I ) D-Ala 2 GIP, immunohistologically stained against Iba1 (scale bar: 80 mum). Bottom half of Figure: effect of D-Ala 2 GIP treatment on three hallmarks of Alzheimer's disease in 12-month-old APP/PS1 mice. ( A ) Quantification of beta-amyloid plaque, ( B ) of dense core plaque load in the cortex of APP/PS1 and, ( C ) of activated microglia (neuroinflammation) in the cor

Supportive validation

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Experimental details: Figure 3. Accumulation of beta-amyloid deposits assessed by immunohistochemistry in E3FAD and E4FAD mice across dietary treatments. A , Representative images of beta-amyloid immunoreactivity in entorhinal cortex and hippocampus in E3FAD and E4FAD males maintained on control and Western diets. Scale bar, 100 um. beta-Amyloid burden was quantified as immunoreactivity load in E3FAD and E4FAD mice in control and Western diets groups in ( B ) entorhinal cortex, and hippocampal subregions ( C ) subiculum, ( D ) CA1, and ( E ) CA2/3. Data are presented as mean (+-SEM) values; n = 7-11/group. E3FAD mice are shown as circles, E4FAD mice are shown as squares; control diet groups are indicated as open symbols, and Western diet groups as filled symbols. *, p < 0.05 relative to genotype-matched mice in control diet condition. #, p < 0.05 relative to E3FAD mice in same diet condition.

Supportive validation

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Experimental details: Figure 1 Distribution of the anti-Abeta antibody and anti-tTG antibodies in sagittal whole brain sections of C57Bl6/J wild-type and APP23 mice brains. 27-months old APP23 mice demonstrated Abeta plaques ( a , black arrow) and vascular Abeta ( a , white arrow). Anti-tTG antibody immunoreactivity was observed in blood vessel walls ( b ) as well as neurons ( b , black arrow) and in glial cells ( b , black open arrow) in both 27-months old wild-type ( b ) and 27-months old APP23 mice (not shown). In APP23 mice, additional tTG antibody immunoreactivity was present in glial cells associated with Abeta plaques ( c-e ) and vascular Abeta ( f-h ) in 24/27-months old mice. Anti-tTG antibody immunoreactivity was associated with the majority of ThioS positive plaques ( i-k ). Double immunofluorescence of the anti-Iba-1 antibody or the anti-GFAP antibody with the anti-tTG antibody demonstrated absence of tTG in Iba-1 positive microglia ( l-n ), whereas tTG immunoreactivity colocalised with GFAP positive astrocytes ( o-q ). Scale bars: ( a ) 20 mum, ( b , f - h , l - q ) 15 mum, ( c - e ) 30 mum, ( i - k ) 60 mum. Abbreviations: Abeta = amyloid beta, GFAP = glial fibrillary acidic protein, Iba-1 = Ionized calcium binding adaptor molecule 1, ThioS = Thioflavin S, tTG = tissue transglutaminase.

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Experimental details: Figure 3 Distribution of in situ TG activity in C57Bl6/J wild-type and APP23 mice. Serial sagittal whole brain sections of wild-type and APP23 mice were incubated with the TG substrate BAP or the anti-Abeta antibody and visualised using the DAB chromogen. The anti-Abeta antibody demonstrated Abeta plaques in 27-months old mice ( b ). BAP staining was found in the cerebral blood vessel walls of both 7-months old wild-type ( a ) and 27-months old APP23 mice ( c ). In addition, in APP23 mice BAP staining was present in Abeta plaque-like structures ( c ). Double immunofluorescence using the anti-Abeta antibody and BAP staining confirmed the presence of TG activity in Abeta plaques in 12-months old mice ( d-f ) as well as in cerebral vessel walls ( e , arrow). BAP staining was found in the majority of dense core plaques, although it was absent from the cores of these plaques, confirmed by double immunofluorescence of ThioS with BAP ( g-i ). Co-incubation of BAP with the selective tTG inhibitor Z-DON (100 muM) blocked the tTG-catalysed incorporation of BAP ( j-l ). Scale bars: ( c , d ) 36 mum, ( a , d - f , j -l) 30 mum, ( g - i ) 15 mum. Abbreviations: Abeta = amyloid beta, BAP = biotinylated 5-(biotinamido)-pentylamine, ThioS = Thioflavin S, tTG = tissue transglutaminase, Z-DON = Z-DON-Val-Pro-Leu-OMe.

Supportive validation

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Experimental details: Figure 4 Distribution of in situ tTG activity in sagittal whole brain sections in C57Bl6/J wild-type and APP23 mice brains. Sagittal serial brain sections of mice were incubated with the specific tTG substrate T26 or the anti-Abeta antibody and visualised using the DAB chromogen. The anti-Abeta antibody stained both plaques ( b ) and vascular Abeta ( d ) in 27-months old mice. T26 staining was present in cerebral blood vessel walls in both 27-months old wild-type ( a ) and APP23 mice ( g , arrow). In addition, in 27-months old APP23 mice, T26 stained both Abeta plaques ( c ) and in vascular Abeta ( e ). Double immunofluorescence of the anti-Abeta antibody with T26 staining demonstrated colocalisation of T26 with Abeta plaques ( f-h ). T26 staining colocalised with the majority of ThioS positive plaques, although T26 staining was absent from the dense cores of these plaques ( i-k ). Co-incubation of T26 with the selective tTG inhibitor Z-DON prevented the tTG-catalysed incorporation of T26 ( l-n ). Scale bars: ( a - c ) 20 mum, ( d - n ) 30 mum. Abbreviations: Abeta = amyloid beta, ThioS = Thioflavin S, tTG = tissue transglutaminase, Z-DON = Z-DON-Val-Pro-Leu-OMe.

Supportive validation

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Experimental details: Figure 5 Quantification of the percentage of anti-Abeta antibody positive and ThioS positive plaques with BAP or T26 staining was performed in 12-months old APP/PS1 mice and 24- and 27-months old APP23 mice. Double immunofluorescence was performed with an anti-Abeta antibody or ThioS with BAP or T26 resulting in double stainings Abeta/BAP, Abeta/T26, ThioS/BAP and ThioS/T26. Only well-defined anti-Abeta antibody positive or ThioS-positive plaques were counted. Well-defined plaques are marked with arrows, both for Abeta ( a ) and ThioS ( b ) staining (APP23 staining shown), whereas examples of plaques that were not taken into quantification are marked with asterisks ( a,b ). The percentages of anti-Abeta antibody positive and ThioS positive plaques positive for BAP or T26 are shown for both APP23 and APP/PS1 mice ( c ). Non-parametric Kruskal-Wallis testing demonstrated a significant higher percentage of ThioS positive plaques with BAP (77.3 +- 1.9% Mean +- SEM) or T26 (73.4 +- 5.2%) staining in APP23 mice compared to APP/PS1 mice where BAP and T26 staining were present in 50.5 +- 8.0% or 38.3 +- 8.1% of the ThioS positive plaques respectively (p = 0.02). In APP23, a trend increase of the percentage of Abeta plaques with T26 staining was present compared to APP/PS1 mice (66.1 +- 6.9% versus 35.5 +- 8.0% respectively, p = 0.06). The increased percentage of Abeta plaques with BAP staining in APP23 mice (61.0 +- 6.9%) compared to APP/PS1 mice (45.7 +- 7.8%) did not reach statisti

Supportive validation

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Experimental details: Figure 8 Abeta deposition in brains of Tg2576 mice at 12 months of age. Coronal brain sections (cortex region) were stained by Congo Red (a) or anti-Abeta polyclonal antibody (b, c). The number of CR-positive plaques on the section was counted in cortex region (a). Typical images of Abeta-immunoreactive tangles of saline-treated Tg2576 mouse (b) and high-dose NK-4(500 ug/kg)-treated Tg2576 mouse (c). Bar represents 50 um.

Supportive validation

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Experimental details: Fig. (5) Representative beta-amyloid (Abeta) immunohistochemistry in 2 (A, B), 5 (C, D) and 13 (E, F) month old 5XFAD mice. Abeta plaque deposition is present in some areas at 2 months of age (A; arrow) and increases with age (C, E). Areas such as the cingulate cortex exhibit intraneuronal Abeta accumulation at 2 months of age (B; arrowheads) which progresses to Abeta plaque deposition at later ages (D, F; open arrowheads). Scale Bars: A, C, E = 500 um; B, D, F = 50 um.

Supportive validation

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Experimental details: FIGURE 5 Measures of Alzheimer-related neuropathology in early- and late-MA female 3xTg-AD mice. Representative pictures of beta-amyloid immunolabeling in the subiculum of the hippocampal formation from each treatment group in (A-D) early-MA and (G-J) late-MA female mice. beta-Amyloid load was measured in the subiculum of (E) early-MA and (K) late-MA. Tau-immunolabeled cells were counted in the subiculum and CA1 hippocampal regions in (F) early-MA and (L) late-MA mice. Significance is p < 0.05. D = significant main effect of diet; H = significant main effect of hormone treatment; X = significant interaction between diet and hormone; d = significant post hoc effect between diets, but within hormone treatment; h = significant post hoc effect between hormone treatment, within diet.

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Experimental details: Figure 4 Analysis of LRP and RAP levels in transgenic animals. Five [APPswe/PS1dE9](+/-)/RAP(+/-) mice (lanes 2, 4, 8, 9), one [APPswe/PS1dE9](-)/RAP(+/+) mouse (lane 1), one [APPswe/PS1dE9](-)/RAP(+/-) mouse (lane 3), one [APPswe/PS1dE9](+/-)RAP(+/+) mouse (lane 5) and two [APPswe/PS1dE9](-)/RAP(-/-) mice (lanes 6, 7) were used in this experiment. 100 ug total protein was loaded per lane. Upper panel - Immunoblot of tissue extract probed with antibody 4109 to RAP (1:1000). Lower panel - Immunoblot of tissue extract probed with antibody 377 to LRP (1:1000). The upper band (~600 kDa) is the full length LRP, the lower band is an 85 kDa fragment of mature, endoproteolytically cleaved, LRP.

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Experimental details: Figure 2 Sex and APOE genotype drive amyloid pathology and microglial gene expression profiles in APOE-FAD mice (A) Representative images of immunohistochemical staining for amyloid-beta (red) and the microglial marker IBA1 (green) in male and female APOE3- and APOE4-FAD mice. Scale bar, 50 mum. (B) Samples separate by APOE and sex along PC1, with APOE3-FAD males clustering separately from mice of both sexes and APOE3-FAD females. A large proportion of the genes driving this separation (76.4%) are DAM-APOE genes. APOE3-FAD males are shown in yellow, APOE3-FAD females in gray, APOE4-FAD males in magenta, and APOE4-FAD females in teal. (C) Heatmap of the DAM-APOE genes associated with sample clustering along PC1. Gene expression levels were normalized, scaled, and centered and are displayed as Z scores. (D and E) Levels of the DAM-APOE marker, CD11c, are increased in APOE3-FAD females and in APOE4-FAD mice of both sexes, both at the (D) transcript and (E) protein levels. (D) Gene expression of Itgax in normalized counts; data are presented as mean (+-SEM) values; n = 5-7/group. *p < 0.05, **p < 0.01; one-way ANOVA with Tukey's multiple comparisons test correction. (E) Representative immunofluorescence images of microglia (IBA1, red) and CD11c (green) in the hippocampal subiculum of male and female APOE3- and APOE4-FAD mice. Scale bar, 25 mum.

Supportive validation

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Experimental details: Figure 5 RI-OR2-TAT reduces the beta-amyloid plaque load and levels of Abeta soluble oligomers in the brains of APP/PS1 transgenic mice. Representative images show amyloid deposits in the cortex region of 10 months old APP/PS1 mouse brains as shown by beta-amyloid immunostaining in animals treated with ( A ) 0.9% saline or ( B ) 100 nmol/kg RI-OR2-TAT in 0.9% saline. ( C ) Quantitative analysis shows a decrease in mean plaque load in the cortex of APP/PS1 mice treated with RI-OR2-TAT compared to animals treated with saline. ( D ) Levels of soluble Ass oligomers in these brains. Values represent mean +- SEM of 4 animals per group, where *** p

Supportive validation

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Experimental details: 5 Fig. 5 Depletion of cytotoxic supernatant using Abeta antibody increases cell killing. (A) PMVECs were incubated with either HBSS, untreated PA103 supernatant, PA103 supernatant incubated with rabbit IgG followed by Protein A agarose beads (IgG Control), or supernatant that was immunodepleted using anti-Abeta or T22. The cells then were photographed at different time points and analyzed. As shown, by 10 h treatment, a time where cytotoxicity had not yet been initiated in untreated control PA103 supernatant, considerable cytotoxic activity was noted in the Abeta depleted supernatant. Bar = 50 mum. (B) Quantitation of cell killing in the five groups shown in Part A at different times following addition of supernatant; dark green = HBSS control, red = PA103 supernatant, yellow = IgG control treated supernatant, light blue = Abeta-depleted supernatant, and light green = T22 (tau)-depleted supernatant ( N = 3).

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Experimental details: 6 Fig. Add back of eluted Abeta to Abeta-depleted supernatant restores cytoprotective characteristics. (A) PMVECs were treated for 10 h with either HBSS, untreated PA103 supernatant, supernatant that was immunodepleted using Abeta antibody, or Abeta-treated supernatant which was reconstituted with proteins that were eluted from protein A agarose beads used for immunodepletion. Bar = 50 mum. (B) Quantitation of cell killing at the 10 h time point in the four groups shown in Part A, and presented as +- SEM. Data were analyzed by one-way ANOVA followed by Tukey's post hoc analysis. N = 3, * P value of 0.002 compared to untreated PA103 supernatant.

Supportive validation

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Experimental details: 7 Fig. (A) Mass spectrometry of PA103 supernatant identified three potential regulators of beta amyloid toxicity. Identified peptides are highlighted. (B) Immunodepletion of cystatin C increases cytotoxicity of PA103 supernatant. PA103 supernatant was immunodepleted with antibodies against either cystatin C (4), gelsolin (5), or Apo J/clusterin (6). The depleted supernatants were then added to PMVECs and cells killing was assessed at 7 h after addition. HBSS (1), PA103 untreated supernatant (2), and Abeta-depleted supernatant (3) were used as controls. Heightened cytotoxicity was detected in the cystatin C-depleted supernatant. Bar = 50 mum. (C) Add back of isolated cystatin C to cystatin C-depleted supernatant partially rescues cytotoxic characteristics of PA103 supernatant. PMVECs were treated with either supernatant that was immune-depleted of cystatin C (1), untreated control PA103 supernatant (2), or anti-cystatin C-treated supernatant that was reconstituted with proteins that were eluted from agarose beads used for immunoprecipitation (3). Bar = 50 mum. (D) Direct quantitation of cell killing at 7 h after addition of control and immunodepleted supernatants, as well as a cystatin C-depleted supernatant that was reconstituted with cystatin C that was eluted from beads following immunodepletion (Add). HBSS and untreated PA103 supernatant served as controls. Data were analyzed by one-way ANOVA followed by Tukey's post hoc analysis. N = 3, +-SEM. * P value of 0.028 compared

Supportive validation

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Experimental details: 2 Fig. Analysis of Proteinase K-treated PA103 supernatant. (A) Supernatant obtained from PA103 infected PMVECs was treated with 100 ug*mL -1 Proteinase K for 0 (untreated control), 1, 4, 8 or 30 h and then added to PMVECs for 21 h. HBSS was added to one group of cells as a negative control. Cell killing was quantified as described, and presented as standard error of the mean (SEM). Data were analyzed by one-way ANOVA followed by Tukey's post hoc analysis. N = 3, * P values compared to PA103 control are 0.005, 0.008, and 0.002 for the 4, 8, and 30 h time points, respectively. (B) Immunoblot analyses of Proteinase K-treated supernatant using antibodies specific for Abeta (MOAb) and oligomeric tau (T22). Protease treatments were for either 1, 4, 8 or 30 h. Untreated PA103 supernatant (PA) was included as a control. Molecular weights are in kDa.

Supportive validation

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Experimental details: 8 Fig. 8 Cystatin C is complexed with Abeta. (A) Co-immunoprecipitation of cystatin C and Abeta was assessed. Supernatant was collected from control cells that were cultured in HBSS for 4 h (Uninfected) and for cells that were infected with PA103 bacteria for 4 h (Infected). The supernatants were precipitated with either cystatin C antibody or control rabbit IgG. The samples were then analyzed by immunoblotting using MOAB anti-Abeta antibody. (B) Both Abeta and cystatin C were detected in pellets following ultracentrifugation of PA103 supernatant. Supernatants were collected either immediately after addition of PA103 bacteria (0 h) or after treatment with bacteria for 2 or 4 h. Each supernatant was centrifuged at 150 000 g for 1, 2, or 4 h and the pelleted material was analyzed using antibody against cystatin C and Abeta (MOAB antibody). Molecular weights are given in kDa.