(10 μg/ml igg) anti-aβ42 rabbit polyclonal antibody ab5078p  (Millipore)

Journal: Acta Neuropathologica Communications

Article Title: A central role for dityrosine crosslinking of Amyloid-β in Alzheimer’s disease

doi: 10.1186/2051-5960-1-83

Figure Lengend Snippet: Formation of dityrosine crosslinks in A β 42 fibrils. Preformed Aβ42 fibrils (20 μM) were incubated in the presence of Cu 2+ /H 2 O 2 for 72 hours and the appearance of dityrosine detected using fluorescence (a) . b) The dityrosine content was confirmed using LC-ESIMS/MS and this shown with relative abundance on y-axis. i) LC-ESIMS/MS from authentic synthetic dityrosine, ii) hydrolysate from oxidized preformed Aβ42 fibrils, iii) hydrolysate from Aβ42 fibrils formed under oxidation conditions for three days (the fibrils were obtained from incubation of soluble Aβ42 with Cu 2+ /H 2 O 2 in water at 37°C and agitation) (c, d) Electron micrographs showing the morphology of fibrils prior to oxidation (c) and following 24 hour oxidation (d) (diameters approximately 100–150 Å).

Article Snippet: In turn, grids were labeled with (10 μg/ml IgG) anti-dityrosine mouse monoclonal antibody (Japan Institute for the Control of Aging JaICA, Shizuoka, Japan) or double-labeled using a mixture of (10 μg/ml IgG) anti-Aβ42 rabbit polyclonal antibody AB5078P (Chemicon, Temecula, CA, USA) and (10 μg/ml IgG) anti-dityrosine mouse monoclonal antibody and incubated overnight at 4°C.

Techniques: Incubation, Fluorescence

Journal: Acta Neuropathologica Communications

Article Title: A central role for dityrosine crosslinking of Amyloid-β in Alzheimer’s disease

doi: 10.1186/2051-5960-1-83

Figure Lengend Snippet: Monitoring dityrosine and tyrosine fluorescence during Aβ42 assembly. Freshly prepared Aβ42 (20 μM) was incubated in the presence of Cu 2+ /H 2 O 2 and monitored by fluorescence over three days (a) and compared to Aβ42 alone (b) . Dityrosine fluorescence was monitored (Ex. 320 nm, Em. 410–420 nm) and a) oxidized Aβ42 shows a strong signal at 24 hours incubation compared to no signal in b) control Aβ42 sample. The development of the dityrosine signal was monitored at earlier time points (c) using the addition of EDTA and the spectrum shows a signal at 420 nm following only 10 mins incubation. Tyrosine fluorescence (Ex. 280 nm, Em. 305 nm) was used to follow assembly with time (d) . The intensity at 305 nm is plotted against time. Both conditions show a decrease in fluorescence signal over time for tyrosine, but oxidized Aβ42 shows a significant reduction after 24 hours, compared to a slow reduction in tyrosine fluorescence that accompanies assembly for control Aβ42.

Article Snippet: In turn, grids were labeled with (10 μg/ml IgG) anti-dityrosine mouse monoclonal antibody (Japan Institute for the Control of Aging JaICA, Shizuoka, Japan) or double-labeled using a mixture of (10 μg/ml IgG) anti-Aβ42 rabbit polyclonal antibody AB5078P (Chemicon, Temecula, CA, USA) and (10 μg/ml IgG) anti-dityrosine mouse monoclonal antibody and incubated overnight at 4°C.

Techniques: Fluorescence, Incubation

Journal: Acta Neuropathologica Communications

Article Title: A central role for dityrosine crosslinking of Amyloid-β in Alzheimer’s disease

doi: 10.1186/2051-5960-1-83

Figure Lengend Snippet: Thioflavine T fluorescence and SDS PAGE of oxidized and control Aβ42. a) ThT fluorescence spectra following fibril formation from Aβ42 (20 μM) in the presence or absence of Cu 2+ and H 2 O 2 for 72 hours. The spectra for oxidized and control samples show an increased intensity over the incubation time and show increased ThT signal for non-oxidized compared to oxidized samples. b) SDS PAGE showing separation of Aβ42. Oxidized Aβ42 (left column) runs as monomer and dimer (approx. 9 kDa, black arrow), whilst non oxidized, control Aβ42 (right column) shows bands corresponding to monomer and trimer as previously observed . The trimer is thought to be induced by SDS . Densitometry confirms that monomer is the strongest band following by dimer for oxidized but not control fibrils. This reveals that the dimer is enriched under oxidation conditions.

Article Snippet: In turn, grids were labeled with (10 μg/ml IgG) anti-dityrosine mouse monoclonal antibody (Japan Institute for the Control of Aging JaICA, Shizuoka, Japan) or double-labeled using a mixture of (10 μg/ml IgG) anti-Aβ42 rabbit polyclonal antibody AB5078P (Chemicon, Temecula, CA, USA) and (10 μg/ml IgG) anti-dityrosine mouse monoclonal antibody and incubated overnight at 4°C.

Techniques: Fluorescence, SDS Page, Incubation

Journal: Acta Neuropathologica Communications

Article Title: A central role for dityrosine crosslinking of Amyloid-β in Alzheimer’s disease

doi: 10.1186/2051-5960-1-83

Figure Lengend Snippet: Transmission electron microscopy images of freshly formed Aβ42 fibrils. 20 μM Aβ42 (pH 7.4) was incubated in the presence of Cu 2+ /H 2 O 2 (a, b, c) and compared to 20 μM Aβ42 in phosphate buffer alone (pH 7.4) (d, e, f) . Assembly was monitored by electron microscopy after incubation for (a and d) zero hours, (b and e) 24 hours and (c and f) 48 hours. Both oxidized and control Aβ42 samples show oligomeric species at zero hour (a, d) and fibrils following 48 hour incubation (c, g) . However, at 24 hours (b and f) fibrils were observed in non-oxidized conditions (f) , but no fibrils were observed in oxidised conditions (b) . The presence of dityrosine was detected using immunogold labeling using a dityrosine specific antibody that labeled oxidized fibrils (g) but not control fibrils (h) .

Article Snippet: In turn, grids were labeled with (10 μg/ml IgG) anti-dityrosine mouse monoclonal antibody (Japan Institute for the Control of Aging JaICA, Shizuoka, Japan) or double-labeled using a mixture of (10 μg/ml IgG) anti-Aβ42 rabbit polyclonal antibody AB5078P (Chemicon, Temecula, CA, USA) and (10 μg/ml IgG) anti-dityrosine mouse monoclonal antibody and incubated overnight at 4°C.

Techniques: Transmission Assay, Electron Microscopy, Incubation, Labeling

Journal: Acta Neuropathologica Communications

Article Title: A central role for dityrosine crosslinking of Amyloid-β in Alzheimer’s disease

doi: 10.1186/2051-5960-1-83

Figure Lengend Snippet: The stability of crosslinked fibrils. a) Oxidized and non-oxidized Aβ42 fibrils were examined using dityrosine fluorescence after prolonged incubation at −80°C showing a strong intensity signal at 420 nm for oxidized but not non-oxidized fibrils. b) Formic acid was used to dissolve the fibrils and the concentration of Aβ42 in solution was compared before and after formic acid dissolution for oxidized and non-oxidized fibrils. c) Electron micrographs of oxidized fibrils before and after formic acid treatment showing that the dityrosine crosslinked fibrils are resistant to formic acid. d) Electron micrographs of non-oxidized fibrils before and after formic acid treatment showing the fibrils are susceptible to damage by formic acid. Scale bars represent 0.2 μm.

Article Snippet: In turn, grids were labeled with (10 μg/ml IgG) anti-dityrosine mouse monoclonal antibody (Japan Institute for the Control of Aging JaICA, Shizuoka, Japan) or double-labeled using a mixture of (10 μg/ml IgG) anti-Aβ42 rabbit polyclonal antibody AB5078P (Chemicon, Temecula, CA, USA) and (10 μg/ml IgG) anti-dityrosine mouse monoclonal antibody and incubated overnight at 4°C.

Techniques: Fluorescence, Incubation, Concentration Assay

Journal: Acta Neuropathologica Communications

Article Title: A central role for dityrosine crosslinking of Amyloid-β in Alzheimer’s disease

doi: 10.1186/2051-5960-1-83

Figure Lengend Snippet: Immunogold labeling TEM showing neuroblastoma cells treated with 10 μM oligomeric Aβ. a) The images reveal dityrosine (10 nm) and Aβ42 (5 nm) labeling within the lysosomes of treated cells. b) low level dityrosine labeling was observed within lysosomes in vehicle treated, control cells, but no Aβ labeling was observed. c) Cells were observed containing fibrillar Aβ labeled for both Aβ (5 nm) and dityrosine (10 nm). White arrows are used to highlight the fibrillar material. Inserts show magnified images for further clarity.

Article Snippet: In turn, grids were labeled with (10 μg/ml IgG) anti-dityrosine mouse monoclonal antibody (Japan Institute for the Control of Aging JaICA, Shizuoka, Japan) or double-labeled using a mixture of (10 μg/ml IgG) anti-Aβ42 rabbit polyclonal antibody AB5078P (Chemicon, Temecula, CA, USA) and (10 μg/ml IgG) anti-dityrosine mouse monoclonal antibody and incubated overnight at 4°C.

Techniques: Labeling

Journal: Acta Neuropathologica Communications

Article Title: A central role for dityrosine crosslinking of Amyloid-β in Alzheimer’s disease

doi: 10.1186/2051-5960-1-83

Figure Lengend Snippet: Electron micrographs of sections of Aβ42 treated neuroblastoma cells showing immunogold labeling of Aβ (5 nm) and dityrosine (10 nm). The images reveal colabeled fibrils outside and also inside the cells and highlight internalization at the plasma membrane (top left panel). The figure shows magnified images in the bottom two panels for extra clarity.

Article Snippet: In turn, grids were labeled with (10 μg/ml IgG) anti-dityrosine mouse monoclonal antibody (Japan Institute for the Control of Aging JaICA, Shizuoka, Japan) or double-labeled using a mixture of (10 μg/ml IgG) anti-Aβ42 rabbit polyclonal antibody AB5078P (Chemicon, Temecula, CA, USA) and (10 μg/ml IgG) anti-dityrosine mouse monoclonal antibody and incubated overnight at 4°C.

Techniques: Labeling

Journal: Acta neuropathologica

Article Title: Frontal cortex and striatal cellular and molecular pathobiology in individuals with Down syndrome with and without dementia

doi: 10.1007/s00401-019-01965-6

Figure Lengend Snippet: List of antibodies

Article Snippet: To validate custom-designed microarray expression data, additional FC sections from both groups were incubated with a rabbit polyclonal antibody against the human nuclear sirtuin 6 (SIRT 6; 1:5000, Sigma), after performing antigen retrieval using Dako target retrieval solution, pH 9 (DAKO, Carpinteria, CA) for 20 minutes in a steamer [ 96 ]. table ft1 table-wrap mode="anchored" t5 Table 2. caption a7 Antigen Antibody Dilution IH (IF) Company Catalog # APP/Aβ (6E10) IgG1 mouse monoclonal to residues 1–16 of Aβ 1:1000 BioLegend #803002 Aβ (MOAB2) IgG2b mouse monoclonal to Aβ 1:800 Gift from M. J. LaDu Aβ42 IgG rabbit polyclonal to Aβ42 1:1000 Millipore #AB5078 Conformational Tau (Alz50) IgM mouse monoclonal to tau residues 5–15, 312–322 1:1000 (1:50) Gift from P. Davies Conformational Tau (MC1) IgG1 mouse monoclonal to tau residues 5–15, 312–322 1:1000 Gift from P. Davies Phosphorylated Tau (pS422) IgG rabbit polyclonal to tau phospho-Serine 422 1:1000 (1:50) Invitrogen # 03151 Phosphorylated Tau (AT8) IgG1 mouse monoclonal to tau phospo-Serine 202 and phospho-Threonine 205 1:1000 (1:50) Invitrogen # MN1020 Truncated Tau (TauC3) IgG1 mouse monoclonal to truncated tau at 421/422 residues 1:1000 (1:50) ThermoFisher # AHB0061 Truncated Tau (MN423) IgG1 mouse monoclonal to truncated tau at glutamic acid 391 1:5000 Gift from L. Binder Choline Acetyltransferase (ChAT) IgG goat polyclonal to human placental choline acetyltransferase 1:800 (1:50) Millipore #AB144P Tyrosine Hydroxylase (TH) IgG rabbit polyclonal to tyrosine hydroxylase 1:1000 Invitrogen #OPA1–04050 Neurofilament (SMI-34) IgG1 mouse monoclonal to phosphorylated neurofilament H 1:1000 BioLegend #8535503 Sirtuin 6 IgG rabbit polyclonal to amino acids 330–348 of human Sirt6 1:5000 Sigma #S4197 Open in a separate window IH, Immunohistochemistry; IF, Immunofluorescence List of antibodies To visualize the relationship between amyloid plaques and tau-containing neurites, additional cortical and striatal sections were dual immunolabeled using two different colored chromogens.

Techniques:

Journal: Acta neuropathologica

Article Title: Frontal cortex and striatal cellular and molecular pathobiology in individuals with Down syndrome with and without dementia

doi: 10.1007/s00401-019-01965-6

Figure Lengend Snippet: Photomicrographs of dual labeled frontal cortex sections showing dystrophic neurites displaying immunoreactivity for the tau conformational epitope MC1 (brown) intermingled within Aβ42-ir plaques (blue) in a 47 year-old female non-demented (a) and a 46 year-old male demented (d) individual with DS. Note the presence of numerous MC1-ir NTs (small arrows) in the demented compared to non-demented DS case. b, c and e, High-power images of plaques (large arrows in a and d) displaying dystrophic neurites. Images of frontal cortex showing Aβ42-ir plaques (blue) surrounded by dystrophic neurites positive for the phosphorylated neurofilament H marker SMI-34 (dark brown) in a 46 year-old male demented person with DS (f). g and h, High-power images showing the bulbous nature of the dystrophic neurites within Aβ42-ir plaques shown in panel f (arrows). Photomicrographs of the putamen showing Aβ42-ir plaques (dark blue) and neuropil lacking MC1-ir dystrophic neurites (brown) in a 47 year-old female non-demented individual with DS (i). Inset shows high-power image of Aβ42-ir plaque from panel i (arrow). j. Image of a dual labeled caudate section demonstrating the absence of SMI-34-ir (dark brown) dystrophic neurites within Aβ42-ir plaques (blue) in a 46 year-old male demented subject with DS. Inset show an apparent intact SMI-34-ir fibers (arrow) in close proximity to an Aβ42-ir plaque (j). k and l, Images showing Aβ42-ir plaques (dark blue) and ChAT-ir neurons (brown) (k) as well as APP/Aβ-ir plaques (blue) and TH-ir fibers (brown) (l) in the putamen of a 46 year-old male demented subject with DS. Note the lack of ChAT-ir (k) and TH-ir (l) dystrophic neurites within plaques. Insets in k and l images show the absence of morphological alterations in a ChAT positive interneuron adjacent to an Aβ42-ir plaque as well as TH-ir fibers neighboring an APP/Aβ-ir plaque indicated (arrows), respectively. Abbreviations, DSD−, DS without dementia, DSD+, DS with dementia. Scale bars in a, d, f and i-l = 50 μm and c, e, g, h and insets =10 μm.

Article Snippet: To validate custom-designed microarray expression data, additional FC sections from both groups were incubated with a rabbit polyclonal antibody against the human nuclear sirtuin 6 (SIRT 6; 1:5000, Sigma), after performing antigen retrieval using Dako target retrieval solution, pH 9 (DAKO, Carpinteria, CA) for 20 minutes in a steamer [ 96 ]. table ft1 table-wrap mode="anchored" t5 Table 2. caption a7 Antigen Antibody Dilution IH (IF) Company Catalog # APP/Aβ (6E10) IgG1 mouse monoclonal to residues 1–16 of Aβ 1:1000 BioLegend #803002 Aβ (MOAB2) IgG2b mouse monoclonal to Aβ 1:800 Gift from M. J. LaDu Aβ42 IgG rabbit polyclonal to Aβ42 1:1000 Millipore #AB5078 Conformational Tau (Alz50) IgM mouse monoclonal to tau residues 5–15, 312–322 1:1000 (1:50) Gift from P. Davies Conformational Tau (MC1) IgG1 mouse monoclonal to tau residues 5–15, 312–322 1:1000 Gift from P. Davies Phosphorylated Tau (pS422) IgG rabbit polyclonal to tau phospho-Serine 422 1:1000 (1:50) Invitrogen # 03151 Phosphorylated Tau (AT8) IgG1 mouse monoclonal to tau phospo-Serine 202 and phospho-Threonine 205 1:1000 (1:50) Invitrogen # MN1020 Truncated Tau (TauC3) IgG1 mouse monoclonal to truncated tau at 421/422 residues 1:1000 (1:50) ThermoFisher # AHB0061 Truncated Tau (MN423) IgG1 mouse monoclonal to truncated tau at glutamic acid 391 1:5000 Gift from L. Binder Choline Acetyltransferase (ChAT) IgG goat polyclonal to human placental choline acetyltransferase 1:800 (1:50) Millipore #AB144P Tyrosine Hydroxylase (TH) IgG rabbit polyclonal to tyrosine hydroxylase 1:1000 Invitrogen #OPA1–04050 Neurofilament (SMI-34) IgG1 mouse monoclonal to phosphorylated neurofilament H 1:1000 BioLegend #8535503 Sirtuin 6 IgG rabbit polyclonal to amino acids 330–348 of human Sirt6 1:5000 Sigma #S4197 Open in a separate window IH, Immunohistochemistry; IF, Immunofluorescence List of antibodies To visualize the relationship between amyloid plaques and tau-containing neurites, additional cortical and striatal sections were dual immunolabeled using two different colored chromogens.

Techniques: Labeling, Marker