E0464

Manufactured by Agilent Technologies

Sourced in Denmark

The E0464 is a laboratory equipment product from Agilent Technologies. It is a precision instrument designed for performing various analytical and experimental tasks in a laboratory setting. The core function of the E0464 is to provide accurate and reliable measurements or data collection, but a detailed description of its specific capabilities or intended use is not available.

Automatically generated - may contain errors

Lab products found in correlation

Streptavidin biotin complex kit, by Vector Laboratories (4 mentions) E0466, by Agilent Technologies (2 mentions) A2547, by Merck Group (1 mentions) Mca497, by Bio-Rad (1 mentions) A4503, by Merck Group (1 mentions) A11058, by Thermo Fisher Scientific (1 mentions) A11059, by Thermo Fisher Scientific (1 mentions) P0397, by Agilent Technologies (1 mentions) Alexa fluor 594 labeled goat anti rat igg, by Thermo Fisher Scientific (1 mentions) Anti f4 80 mca497ga, by Bio-Rad (1 mentions) Ab15627, by Abcam (1 mentions) K3467, by Agilent Technologies (1 mentions) Ab5694, by Abcam (1 mentions) Chemidoc xr, by Bio-Rad (1 mentions) Image lab, by Bio-Rad (1 mentions) Hybond c, by Cytiva (1 mentions) Hybond, by Cytiva (1 mentions) Polyclonal goat anti rabbit, by Agilent Technologies (1 mentions) F0232, by Agilent Technologies (1 mentions) Zuc028 500, by Zytomed Systems (1 mentions)

10 protocols using e0464

Immunohistochemical Detection of Myofibroblasts and Macrophages

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A mouse monoclonal anti-α-SMA antibody (A-2547, Sigma-Aldrich) and a mouse monoclonal anti-F4/80 antibody (MCA497, Serotec) were used to detect myofibroblasts and macrophages, respectively. Sections cut at 4 µm were dewaxed, incubated in 1.8% v/v hydrogen peroxide in methanol for 15 min to block endogenous peroxidases, and then taken through graded alcohols to PBS. The sections were then incubated in 20% v/v acetic acid in methanol to block endogenous alkaline phosphatases.
Antigen retrieval treatment was performed by microwaving sections in BD Retrievagen A solution (550524, BD Pharmagen) for 10 minutes. To reduce nonspecific background staining, sections were next preincubated with 1% bovine serum albumin (A4503, Sigma-Aldrich) for 30 minutes. Primary antibodies were used at dilutions of 1 : 4000 for α-SMA and 1 : 100 for F4/80. The secondary antibody for α-SMA and F4/80 was a biotinylated polyclonal rabbit anti-mouse immunoglobulin used at a dilution of 1 : 300 (E0464, Dako). The tertiary layer was alkaline phosphatase-conjugated streptavidin (D0396; Dako) used at a 1 : 50 dilution. Slides were further developed in DAB and counterstained lightly with haematoxylin, dehydrated and mounted in DPX-type mount.

Lin W.R., Lim S.N., Yen T.H, & Alison M.R. (2016). The Influence of Bone Marrow-Secreted IL-10 in a Mouse Model of Cerulein-Induced Pancreatic Fibrosis. BioMed Research International, 2016, 4601532.

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Immunohistochemical Analysis of AAV-PD Model

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To pilot the rationale of a VEP study in the AAV model of PD, immunohistochemistry was performed on four perfusion fixed brains from a previous study of the AAV-model in our lab. The brains were extracted at week 10–11 after injection, and cut in slices of 40 µm on a freeze microtome and placed in potassium phosphate buffered saline (KPBS). The tissue was quenched with hydrogen peroxide and washed in KPBS before being incubated with primary antibody hWT-α-synuclein (4B12) (Thermo scientific, US) in the concentration 1:1,000. Tissue was then washed before being incubated with the secondary antibody (E0464, DAKO, Denmark) and exposed using a 3,3-diaminobenzidine-reaction.

Østergaard F.G., Himmelberg M.M., Laursen B., Siebner H.R., Wade A.R, & Christensen K.V. (2020). Classification of α-synuclein-induced changes in the AAV α-synuclein rat model of Parkinson’s disease using electrophysiological measurements of visual processing. Scientific Reports, 10, 11869.

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Immunohistochemical Analysis of Tau Pathology

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Example 6

Mouse brain tissues were collected from 8 months old rTg4510 mice (overexpressing human P301L-tau under the CamKII promoter) and non-transgenic littermate (non-Tg), fixed in 4% paraformaldehyde and embedded in paraffin. Paraffin-embedded human brain samples of frontal cortex were acquired from Tissue Solutions (Glasgow, UK). Tissue from donors with diagnosed end stage Alzheimer's disease was compared to age-matched non-demented control donors. Four um thick sections were deparaffinized and subjected to antigen retrieval by microwaving the sections in 10 mM Citrate buffer, pH 6, for 10 minutes. Endogenous peroxidases were blocked with 1% hydrogen peroxidase followed by 5% normal swine serum in PBS/1% BSA/0.3% Triton X-100 (PBS-BT). Sections were incubated overnight at 4° C. with D1.2 and C10-2 antibodies diluted in PBS-BT at a range of concentrations. The sections were washed in PBS, 0.25% BSA, 0.1% Triton X-100, before being incubated with a biotinylated secondary swine anti-mouse antibody (E0464; DAKO, Glostrup, Denmark) at 1:500 for 1 hour. Following additional washing, StreptAvidin-Biotin Complex kit (Vector Laboratories, Burlingame, Calif.) was applied and immunoreactivity was visualized with diaminobenzidine. Sections were counterstained with hematoxylin. Results are shown in FIG. 5.

US10562962B2. Antibodies specific for hyperphosphorylated tau and methods of use thereof (2020-02-18). H. Lundbeck A/S [DK]. Inventors: Jan Torleif Pedersen [DK], Lars Østergaard Pedersen [DK], Justus Claus Daechsel [DK], Ayodeji Abdur-rasheed Asuni [DK], Nina Helen Rosenqvist [DK], Christiane Volbracht [DK], Lone Helboe [DK], Anders Brandt Elvang [DK], Florence Sotty [DK], Søren Christensen [DK], Jeffrey B. Stavenhagen [DK].

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Tau Protein Immunohistochemistry in Alzheimer's

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Example 6

Mouse brain tissues were collected from 8 months old rTg4510 mice (overexpressing human P301L-tau under the Cam KII promoter) and non-transgenic littermate (non-Tg), fixed in 4% paraformaldehyde and embedded in paraffin. Paraffin-embedded human brain samples of frontal cortex were acquired from Tissue Solutions (Glasgow, UK). Tissue from donors with diagnosed end stage Alzheimer's disease was compared to age-matched non-demented control donors. Four um thick sections were deparaffinized and subjected to antigen retrieval by microwaving the sections in 10 mM Citrate buffer, pH 6, for 10 minutes. Endogenous peroxidases were blocked with 1% hydrogen peroxidase followed by 5% normal swine serum in PBS/1% BSA/0.3% Triton X-100 (PBS-BT). Sections were incubated overnight at 4° C. with D1.2 and C10-2 antibodies diluted in PBS-BT at a range of concentrations. The sections were washed in PBS, 0.25% BSA, 0.1% Triton X-100, before being incubated with a biotinylated secondary swine anti-mouse antibody (E0464; DAKO, Glostrup, Denmark) at 1:500 for 1 hour. Following additional washing, StreptAvidin-Biotin Complex kit (Vector Laboratories, Burlingame, Calif.) was applied and immunoreactivity was visualized with diaminobenzidine. Sections were counterstained with hematoxylin. Results are shown in FIG. 5.

US10196439B2. Antibodies specific for hyperphosphorylated tau and methods of use thereof (2019-02-05). H. Lundbeck A/S [DK]. Inventors: Jan Torleif Pedersen [DK], Lars Østergaard Pedersen [DK], Justus Claus Alfred Daechsel [DE], Ayodeji Abdur-Rasheed Asuni [DK], Nina Helen Rosenqvist [SE], Christiane Volbracht [DK], Lone Helboe [DK], Anders Brandt Elvang [DK], Florence Sotty [DK], Søren Christensen [DK], Jeffrey B. Stavenhagen [DK].

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Tau Pathology in Alzheimer's and rTg4510 Mice

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Example 6

US11739140B2. Antibodies specific for hyperphosphorylated tau and methods of use thereof (2023-08-29). H. Lundbeck A/S [DK]. Inventors: Jan Torleif Pedersen [DK], Lars Østergaard Pedersen [DK], Justus Claus Alfred Daechsel [DK], Ayodeji Abdur-rasheed Asuni [DK], Nina Helen Rosenqvist [DK], Christiane Volbracht [DK], Lone Helboe [DK], Anders Brandt Elvang [DK], Florence Sotty [DK], Søren Christensen [DK], Jeffrey B. Stavenhagen [DK].

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Immunohistochemical Analysis of Liver Markers

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Liver sections were incubated with primary antibodies, followed by incubation with biotinylated secondary antibodies. The following primary antibodies were used: anti-STK25 (YSK1; sc-6865; Santa Cruz Biotechnology, Santa Cruz, CA), anti-PCNA (MA5-1158; Invitrogen), anti-Ki67 (14-5698-82; Invitrogen), anti-Gr1 (Ly6C) (ab15627; Abcam, Cambridge, UK), anti-F4/80 (MCA497GA; Bio-Rad, Hercules, CA), anti–collagen I (SAB4500362; Sigma-Aldrich), anti–α-smooth muscle actin (ab5694; Abcam), anti–4-HNE (sc-130083; Santa Cruz Biotechnology), anti-PEX5 (PA5-58716; Invitrogen), and anti-ubiquitin (ab411; Abcam). For immunohistochemical detection, anti-goat IgG (E0466; Dako, Glostrup, Denmark) and anti-mouse IgG (E0464; Dako) secondary antibodies were used, followed by horseradish-peroxidase–conjugated streptavidin (P0397; Dako) and diaminobenzidine staining (K3467; Dako). For immunofluorescence detection, Alexa Fluor-594–labeled goat anti-rat IgG (A11007; Invitrogen), Alexa Fluor-488–labeled rabbit anti-mouse IgG (A11059; Invitrogen), Alexa Fluor-594–labeled donkey anti-goat IgG (A11058; Invitrogen), and Alexa Fluor-594–labeled donkey anti-rabbit IgG (A21207; Invitrogen) secondary antibodies were used. The stained area was quantified in 5 randomly selected microscopic fields (×200) per mouse using ImageJ software.

Cansby E., Nuñez-Durán E., Magnusson E., Amrutkar M., Booten S.L., Kulkarni N.M., Svensson L.T., Borén J., Marschall H.U., Aghajan M, & Mahlapuu M. (2018). Targeted Delivery of Stk25 Antisense Oligonucleotides to Hepatocytes Protects Mice Against Nonalcoholic Fatty Liver Disease. Cellular and Molecular Gastroenterology and Hepatology, 7(3), 597-618.

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Immunohistochemical Analysis of Tau Pathology

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Example 6

Mouse brain tissues were collected from 8 months old rTg4510 mice (overexpressing human P301L-tau under the CamII promoter) and non-transgenic littermate (non-Tg), fixed in 4% paraformaldehyde and embedded in paraffin. Paraffin-embedded human brain samples of frontal cortex were acquired from Tissue Solutions (Glasgow, UK). Tissue from donors with diagnosed end stage Alzheimer's disease was compared to age-matched non-demented control donors. Four um thick sections were deparaffinized and subjected to antigen retrieval by microwaving the sections in 10 mM Citrate buffer, pH 6, for 10 minutes. Endogenous peroxidases were blocked with 1% hydrogen peroxidase followed by 5% normal swine serum in PBS/1% BSA/0.3% Triton X-100 (PBS-BT). Sections were incubated overnight at 4° C. with D1.2 and C10-2 antibodies diluted in PBS-BT at a range of concentrations. The sections were washed in PBS, 0.25% BSA, 0.1% Triton X-100, before being incubated with a biotinylated secondary swine anti-mouse antibody (E0464; DAKO, Glostrup, Denmark) at 1:500 for 1 hour. Following additional washing, StreptAvidin-Biotin Complex kit (Vector Laboratories, Burlingame, Calif.) was applied and immunoreactivity was visualized with diaminobenzidine. Sections were counterstained with hematoxylin. Results are shown in FIG. 5.

US10934348B2. Antibodies specific for hyperphosphorylated tau and methods of use thereof (2021-03-02). H. Lundbeck A/S [DK]. Inventors: Jan Torleif Pedersen [DK], Lars Østergaard Pedersen [DK], Justus Claus Alfred Daechsel [DK], Ayodeji Abdur-rasheed Asuni [DK], Nina Helen Rosenqvist [DK], Christiane Volbracht [DK], Lone Helboe [DK], Anders Brandt Elvang [DK], Florence Sotty [DK], Søren Christensen [DK], Jeffrey B. Stavenhagen [DK].

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Western Blot Analysis of MCF-7 Cells

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Forty micrograms of all cellular fractions (MCF-7 cells) were separated using 8 and 10 % SDS-polyacrylamide gel electrophoresis and electrotransferred onto a nitrocellulose membrane (Hybond-C, Amersham biosciences, United Kingdom). After blocking in 5 % milk (wt/vol) in PBS, 0.1 % Tween 20 (vol/vol) membranes were incubated with primary antibodies overnight at 4 °C. Followed by washes whit PBS and then were incubated with secondary antibody. The following antibodies were used: anti b-catenin (cat. no. 18-0226, Zymed, San Francisco CA); anti HER2 (cat. no. 554299, BD Transduction, Lab Pharmingen, San Diego CA), anti a-tubulin (cat. T-6074, Sigma-Aldrich), anti cleaved PARP1 (BD Transduction, Lab Pharmingen, San Diego, CA). The secondary antibodies were horseradish peroxidase-conjugated (E0464, DAKO). Immunodetection was carried out using enhanced chemiluminescence and was recorded with a quantitative digital imaging system (Chemidoc XRS with Image Lab, Bio-Rad, Hercules, CA, USA), enabling us to assess saturation. Band densitometric analysis was performed on some blots using conditions ensuring analysis of signals in the linear range of detection.

Cuello-Carrión F.D., Shortrede J.E., Alvarez-Olmedo D., Cayado-Gutiérrez N., Castro G.N., Zoppino F.C., Guerrero M., Martinis E., Wuilloud R., Gómez N.N., Biaggio V., Orozco J., Gago F.E., Ciocca L.A., Fanelli M.A, & Ciocca D.R. (2015). HER2 and β-catenin protein location: importance in the prognosis of breast cancer patients and their correlation when breast cancer cells suffer stressful situations. Clinical & experimental metastasis, 32(2).

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Antibody Staining of Human Siglecs

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Antibodies against human Siglecs 1, 2, 5, 6, 10 and 14 that recognise extracellular epitopes in regions and domains that are conserved between human and other species were employed and are detailed in Table 1 andSupplementary Table 5A andB (see section on supplementary data given at the end of this article). Antibodies were used at a dilution between 1:35 and 1:200. Positive staining was detected by secondary staining with either FITC-conjugated or biotinylated antibodies. Secondary antibodies employed were polyclonal rabbit anti-mouse (F0232, FITC conjugated), polyclonal rabbit anti-mouse (E0464, biotinylated), polyclonal rabbit anti-goat (F0250, FITC conjugated) and polyclonal rabbit anti-goat (E0466, biotinylated), all from DakoCytomation.

Alkhodair K., Almhanna H., McGetrick J., Gedair S., Gallagher M.E., Fernandez-Fuertes B., Tharmalingam T., Larsen P.B., Fitzpatrick E., Lonergan P., Evans A.C.O., Carrington S.D, & Reid C.J. (2018). Siglec expression on the surface of human, bull and ram sperm. Reproduction (Cambridge, England), 155(4).

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Quantitative Immunohistochemical Analysis of Ki67

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The semipermeable membranes were excised from the TWI and fixed in 4% phosphate-buffered formaldehyde. Antigens were demasked by heating the samples to 97 °C in a steam oven in citric acid buffer (pH 6, Zuc 028-500, Zytomed Systems, Berlin, Germany) for 20 min. Ki67 staining was carried out by mouse anti-human monoclonal Ki67/MIB-1 antibody (M7240, Dako, Glostrup, Denmark) in a 1:50 dilution and incubated at room temperature over night. A polyclonal biotinylated rabbit anti-mouse antibody (E0464, Dako) in a 1:500 dilution was used as secondary antibody for 1 hr. Streptavidin/HRP (Zytomed Systems) and DAB (BD Pharmingen 550880, Becton, Dickinson and Company, Franklin Lakes, NJ, USA) were added to detect the presence of the secondary antibody. Haematoxylin was used for nuclear staining (MHSA 128, Sigma-Aldrich).
All cells and Ki67 cells were counted in three randomly selected microscopic fields measuring 2.25 cm 2 of each TWI by a masked investigator (MF). Every compound was tested in triplets.

Hesse M., Kuerten D., Walter P., Plange N., Johnen S, & Fuest M. (2017). The effect of air, SF6 and C3F8 on immortalized human corneal endothelial cells. Acta ophthalmologica, 95(4).

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