Dako envision dual link system hrp

Manufactured by Agilent Technologies

Sourced in United States

The Dako EnVision+ Dual Link System-HRP is a labeling system used in immunohistochemistry and in situ hybridization techniques. It is designed to detect primary antibodies or nucleic acid probes in tissue sections or cell preparations. The system utilizes a polymer-based detection technology that amplifies the signal, enabling the visualization of target molecules.

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Envision System (550 citations) EnVision+ System-HRP (201 citations) Dako Envision system (132 citations) EnVision+ Dual Link System-HRP (121 citations) EnVision/HRP (63 citations) Dako EnVision System HRP (46 citations) EnVision+ Dual Link System (32 citations) EnVision+ Dual Link (25 citations) Dako EnVisionTM + Dual Link System-HRP (DAB+) (12 citations) Envision+Dual Link HRP (5 citations)

31 protocols using dako envision dual link system hrp

Immunohistochemistry for FOXO1 and PAX3 in FFPE Tissue

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The TMA sections were deparaffinized and rehydrated using xylene and ethanol solutions of descending alcohol gradient. All slides were quenched for 10 min in 3% H₂O₂ to block the endogenous peroxidase. Heat-induced antigen retrieval was performed in an antigen retrieval buffer of pH 9 (Dako, Carpinteria, CA, USA) for FOXO1 and pH 6 (Dako) for PAX3, in a steam pressure cooker for 10 min (Pascal, Dako). The slides were then stained with an anti-FOXO1 antibody (Abcam, Cambridge, MA, USA; rabbit antibody, clone# EP927Y, 1:400) and an anti-PAX3 antibody (Abcam, rabbit polyclonal antibody, Cat.# Ab216683, 1:200) for 1 h at room temperature in a Dako Autostainer Plus slide stainer (Dako). The antigen–antibody reaction was detected with the Dako EnVision + Dual Link System-HRP (Dako) and DAB⁺ (3,3′-diaminobenzidine; Dako).

Chay D.B., Han G.H., Nam S., Cho H., Chung J.Y, & Hewitt S.M. (2019). Forkhead box protein O1 (FOXO1) and paired box gene 3 (PAX3) overexpression is associated with poor prognosis in patients with cervical cancer. International journal of clinical oncology, 24(11), 1429-1439.

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Immunohistochemical Analysis of HIF-1α

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At euthanasia, en bloc extraction of the AVF or the infrarenal aorta + IVC (control) was performed after perfusion-fixation with PBS followed by 10% formalin. The tissue block was then embedded in paraffin and cut into 5µm cross-sections. Immunohistochemistry was performed using the Dako EnVision+ Dual Link System-HRP (Dako, Carpinteria, CA). Sections were heated in citric acid buffer (pH 6.0) at 100°C for 10 min using the Lab Vision PT Module (Thermo Scientific, Kalamazoo, MI) for antigen retrieval, then treated with 0.3% hydrogen peroxide in methanol for 30 min at room temperature to block endogenous peroxidase activity. They were incubated with 5% normal goat serum in PBS (pH 7.4) containing 0.05% Triton X-100 for 1 h at room temperature to block nonspecific protein-binding sites. Sections were then incubated at 4°C with the anti-HIF-1α primary antibody diluted at 1:50 in PBS containing 0.05% Triton X-100. After an overnight incubation, sections were incubated with EnVision reagents for 1 h at room temperature and treated with the Dako Liquid DAB+ Substrate Chromogen System (Dako) to visualize the reaction products. Finally, sections were counterstained with Dako Mayer’s Hematoxylin (Lillie’s Modification) Histological Staining Reagent (Dako).

Sadaghianloo N., Yamamoto K., Bai H., Tsuneki M., Protack C.D., Hall M.R., Declemy S., Hassen-Khodja R., Madri J, & Dardik A. (2017). Increased oxidative stress and hypoxia inducible factor-1 expression during arteriovenous fistula maturation. Annals of vascular surgery, 41, 225-234.

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Immunohistochemical Analysis of CT45 and YBX2

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Paraffin sections were deparaffinized, and rehydrated. Slides were washed in PBS, and immune-labeled with primary antibodies against CT45 and YBX2 overnight at 4 °C after incubation with Protein Block Serum Free (Dako, Carpentaria, CA) to eliminate nonspecific binding. The slides were then rinsed with PBS and incubated with anti-rabbit secondary antibody (DAKO Envision + Dual Link System HRP; Dako) at room temperature for 1 h. Visualization of the immunoreaction was carried out by incubation with 3,3-diaminobenzidine (DAB). Finally, sections were counterstained with hematoxylin. The antibodies used with dilution and providers are listed in Supplementary Table S7 online. Immunohistochemistry staining was detected under the BZ-X710 microscope (Keyence, Osaka, Japan). The entire slide was evaluated with the Allred scoring system^{36 (link)}: two categories (stain intensity and stain pattern) were evaluated (Supplementary Table S2 online).

Suzuki I., Yoshida S., Tabu K., Kusunoki S., Matsumura Y., Izumi H., Asanoma K., Yagi H., Onoyama I., Sonoda K., Kohno K., Taga T., Itakura A., Takeda S, & Kato K. (2021). YBX2 and cancer testis antigen 45 contribute to stemness, chemoresistance and a high degree of malignancy in human endometrial cancer. Scientific Reports, 11, 4220.

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Immunohistochemical Analysis of Vascular Markers

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Tissue sections were de-paraffined using xylene and a graded series of alcohols, non-specific background staining of endogenous peroxidase was treated with 1% hydrogen peroxide for 30 minutes, and sections were blocked with 1% bovine serum albumin prior to incubation with primary antibodies overnight at 4°C. Primary antibodies were directed against the endothelial marker CD31 (ab28364, Abcam; 1:50 dilution), the smooth muscle marker α-actin (ab5694, Abcam; 1:100 dilution), the progenitor marker CD34 (AF4117, R&D; 1:100 dilution), the endothelial marker VEGFR2 (ab2349, Abcam; 1:50 dilution), the M2 macrophage marker anti-transglutaminase 2 (TGM2; #37557; Cell Signaling; 1:50 dilution), proliferating cell nuclear antigen (PCNA; M0879, Dako ; 1:100 dilution) or the apoptosis marker cleaved caspase-3 (#9661; Cell Signaling; 1:100 dilution). Detection was performed using Dako EnVision^™ + Dual Link System-HRP (Dako; Carpinteria, CA), and counterstained with Mayer’s Hematoxylin. The integrated optical density of smooth muscle cells (SMC) in the neointima were analyzed using Image-Pro Plus 6.0 software (Media Cybernetics; Rockville, MD). Cells staining positively for CD34, VEGFR2, PCNA (proliferation index), or cleaved caspase-3 (apoptosis index) were directly counted in 4 high power fields, and compared to the total number of cells in the field.

Bai H., Kuwahara G., Wang M., Brownson K., Foster T., Yamamoto K., Xing Y, & Dardik A. (2014). Pretreatment of pericardial patches with antibiotics does not alter patch healing in vivo. Journal of vascular surgery, 63(4), 1063-1073.

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Immunohistochemical Profiling of Breast Cancer

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The FFPE specimens from the 92 patients were examined. Tissue sections were deparaffinized and hydrated through graded alcohols and xylene. Antigen retrieval was carried out with citrate buffer at pH 6.0 in an autoclave at 121°C for 10 minutes. Endogenous peroxidase was blocked with 3% hydrogen peroxide for 10 minutes. After rinsing and blocking with 5% normal donkey serum, the sections were incubated overnight at 4°C with primary Ab. The sections were washed and incubated for 2 hours at 4°C with the Dako EnVision + Dual Link System‐HRP (Dako). Diaminobenzidine (Dako EnVision kit/HRP [DAB]) was used for detection of protein. The sections were finally counterstained with hematoxylin. On IHC, ER status and PgR status were assessed semiquantitatively and reported as positive when more than 1% of the nuclei of cancer cells showed staining. Positive HER2 status was determined if strong staining of the complete membrane in more than 10% of tumor cells was observed. Details of Abs are as follows: ER, rabbit monoclonal, clone SP1 (Ventana); PgR, rabbit monoclonal, clone 1E2 (Ventana); and HER2, rabbit monoclonal, clone 4B5 (Ventana). For Ki‐67 staining, mAb (MIB‐1; Dako) (1:400) was used and the cells positive for nuclear Ki‐67 were counted in at least 500 cancer cells in one hotspot on each sample.

Murakami F., Tsuboi Y., Takahashi Y., Horimoto Y., Mogushi K., Ito T., Emi M., Matsubara D., Shibata T., Saito M, & Murakami Y. (2020). Short somatic alterations at the site of copy number variation in breast cancer. Cancer Science, 112(1), 444-453.

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Macrophage Quantification in Kidney Tissue

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Immunohistochemistry staining of F4/80, a marker of macrophages, was performed on 4 µm paraffin sections treated by 40 µg/ml proteinase K (Sigma, Dorset, UK) at 37°C, 30 min for antigen retrieving. F4/80 (1:50, ab11101, Abcam, Cambridge, UK) was incubated at 4°C overnight. DAKO EnVision™ + Dual Link System-HRP was used as the secondary antibody (DAKO, Glostrup, Denmark). The antibody binding was revealed by 3,3’-diaminobenzidine (SK-4100, DAB, Vector, Burlingame, USA) with hematoxylin counterstaining. The number of macrophages in the tubular, interstitial, and tubular lumen areas were semi-quantitatively analysed in 20 fields at 400× magnification.

Zhang Y., Wu Y., Wang W., Liu F., Zhang Y., Yang C., Liu A., Wu J., Zhu T., Nicholson M.L., Fan Y, & Yang B. (2021). Long-Term Protection of CHBP Against Combinational Renal Injury Induced by Both Ischemia–Reperfusion and Cyclosporine A in Mice. Frontiers in Immunology, 12, 697751.

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Immunohistochemical Analysis of Lymphocytes

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Mouse spleens and livers were fixed in 10% neutral-buffered formalin and embedded in paraffin. Sections of 10 μm thickness were deparaffinized and antigens were retrieved using citrate-based buffer (pH 6) for 10 min (H3300, Vector Laboratories). Slides were incubated in blocking solution (3% bovine serum albumin + tris-buffered saline with Tween 20) for 1 h and incubated overnight with mouse anti-human CD3 (Dako, Clone F7.2.38, #M725401-2, 1:100) and mouse anti-human CD20cy (Dako, Clone L26, #IR604, 1:50). Slides were then incubated with an HRP-conjugated secondary antibody (Dako EnVision+ Dual Link System-HRP, Dako, #K4063). The slides were developed with diaminobenzidine (ImmPACT DAB Peroxidase (HRP) Substrate, Vector Laboratories, #SK-4105).

Harada T., Kikushige Y., Miyamoto T., Uno K., Niiro H., Kawakami A., Koga T., Akashi K, & Yoshizaki K. (2023). Peripheral helper-T-cell-derived CXCL13 is a crucial pathogenic factor in idiopathic multicentric Castleman disease. Nature Communications, 14, 6959.

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Immunohistochemical Analysis of VEGF Signaling

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Formalin fixed tissue was stained by routine H-E staining or immunohistochemistry (IHC). The following primary antibodies were used for IHC: anti-VEGF-A antibody (Merck Millipore Japan, Tokyo, Japan), anti-VEGFR-1 (R&D Biosystems, Minneapolis, MN, USA), and anti-VEGFR-2 antibody (R&D Biosystems). All primary antibodies were incubated with tissues at a 1:100 dilution for 60 min. The secondary antibody Dako EnVision™+ Dual Link System-HRP (Dako, Tokyo, Japan) was then incubated with the tissues for 15 min at room temperature. Protein expression was visualized using a 3,3′-diaminobenzidine tablet (Dako).

Araki-Maeda H., Kawabe M., Omori Y., Yamanegi K., Yoshida K., Yoshikawa K., Takaoka K., Noguchi K., Nakano Y, & Kishimoto H. (2022). Establishment of an oral squamous cell carcinoma cell line expressing vascular endothelial growth factor a and its two receptors. Journal of Dental Sciences, 17(4), 1471-1479.

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Immunohistochemical Analysis of Vascular Markers

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Sections were heated in citric acid buffer (pH 6.0) at 100°C for 10 min for antigen retrieval. The sections were treated with 0.3% hydrogen peroxide in methanol for 30 min at room temperature to block endogenous peroxidase activity and incubated with 5% normal goat serum in PBS (pH 7.4) containing 0.05% Triton X-100 (T-PBS) for 1 h at room temperature to block nonspecific protein-binding sites. Sections were then incubated at 4°C with the primary antibodies diluted at 1:100 (anti-α-actin), 1:200 (anti-CD31), and 1:100 (anti-Klf2) in T-PBS. After overnight incubation, the sections were incubated with Dako EnVision™ + Dual Link System-HRP (Dako, Carpinteria, CA) or secondary anti-goat antibody (sc-2020; Santa Cruz Biotechnology, Dallas, TX) for 1 h at room temperature and treated with Dako Liquid DAB+ Substrate Chromogen System (Dako) to visualize the reaction products. Finally, the sections were counterstained with Dako Mayer's Hematoxylin (Lillie's Modification) Histological Staining Reagent (Dako).

Yamamoto K., Protack C.D., Kuwahara G., Tsuneki M., Hashimoto T., Hall M.R., Assi R., Brownson K.E., Foster T.R., Bai H., Wang M., Madri J.A, & Dardik A. (2015). Disturbed shear stress reduces Klf2 expression in arterial-venous fistulae in vivo. Physiological Reports, 3(3), e12348.

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Immunohistochemical Analysis of Survivin Expression

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To examine the protein expression of survivin, immunohistochemistry was performed using a tissue array TH641 (US Biomax, Inc) with a rabbit polyclonal antibody (Novus Biologicals, NB500-201) at 1:500 dilution. Briefly, slides were deparaffinized in xylene and graded alcohols, and subjected to antigen retrieval in a pressure cooker with citrate buffer (pH6) for 20 minutes. Endogenous enzyme activity was blocked with 3% hydrogen peroxide in methanol with additional serum-free protein blocking to reduce nonspecific reactions. Subsequently, slides were incubated with primary antibody for 60 minutes at room temperature, and antigen–antibody reaction was detected with Dako Envision+ Dual Link system-HRP (Cat.K4061, Dako), visualized in 3,3'-diaminobenzidine, counterstained with hematoxylin, dehydrated, and coverslipped. The staining result was reviewed by a pathologist (A.R.).

Mehta A., Zhang L., Boufraqech M., Liu-Chittenden Y., Zhang Y., Patel D., Davis S., Rosenberg A., Ylaya K., Aufforth R., Li Z., Shen M, & Kebebew E. (2015). Inhibition of survivin with YM155 induces durable tumor response in anaplastic thyroid cancer. Clinical cancer research : an official journal of the American Association for Cancer Research, 21(18), 4123-4132.

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