Dako envision flex kit

Manufactured by Agilent Technologies

Sourced in Denmark, United States

The Dako Envision Flex Kit is a laboratory equipment product used for immunohistochemistry (IHC) procedures. It provides a detection system for the visualization of antigens in tissue sections or cell preparations.

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Lab products found in correlation

Close spelling variants of this product

Dako EnVision FLEX detection kit (2 citations) Dako EnVision™ FLEX High pH Detection Kit (2 citations) Dako Envision Flex Plus Mouse Link Kit (2 citations) Dako EnVision Flex (10 citations) DAKO Envision kit (90 citations) EnVision FLEX kit (77 citations) EnVision FLEX (142 citations) Dako kits (2 citations) Envision kit (439 citations)

13 protocols using dako envision flex kit

FGFR2 Immunohistochemistry Protocol

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Following deparafinization and rehydration, antigen retrieval was followed by blocking of endogenous peroxidize activity using H₂O₂ block (Dako Envision Flex Kit; K8000).
For FGFR2 IHC, antigen retrieval was done with pressure cooking in pH 9 retrieval buffer (DakoS2367). Sections were rinsed with TBS-Tween 0.05% and endogenous peroxidise blocked using H₂O₂ block (Dako Envision Flex Kit; K8000). Sections were incubated for 1 hour with 1/10,000 FGFR2 antibody (AZ AGG 2935-1C11, mouse monoclonal, from AstraZeneca), followed by Envision+/HRP (Dako; K4001) for 30 minutes and staining with 3,3′-diaminobenzadine (DAB). Sections were washed and counterstained in Gill’s Hematoxylin.

Pearson A., Smyth E., Babina I.S., Herrera-Abreu M.T., Tarazona N., Peckitt C., Kilgour E., Smith N.R., Geh C., Rooney C., Cutts R., Campbell J., Ning J., Fenwick K., Swain A., Brown G., Chua S., Thomas A., Johnston S.R., Ajaz M., Sumpter K., Gillbanks A., Watkins D., Chau I., Popat S., Cunningham D, & Turner N.C. (2016). High-Level Clonal FGFR Amplification and Response to FGFR Inhibition in a Translational Clinical Trial. Cancer discovery, 6(8), 838-851.

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Immunohistochemical Analysis of HIF-1α and CAIX

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Immunohistochemistry was performed on representative tissue block sections, chosen using hematoxylin and eosin stainings. Commercial monoclonal mouse antibody against HIF-1alpha (NB100-105 IgG2b, Clone H1alpha67, Novus Biologicals, Littleton, CO) has previously been validated for use in formalin-fixed paraffin-embedded material [24 (link)–26 (link)] and was used at a dilution of 1:300. Dako Envision flex kit (Dako, Copenhagen, Denmark) with high-temperature antigen retrieval in Tris-EDTA buffer for 20 min (pH 9.0) was used for detection of antibody reaction and diaminobenzidine (Dako basic DAB-kit) as a chromogen. Dako Autostainer (Dako) was used for the stainings.
The previously described monoclonal antibody M75 was used to recognize the N-terminal domain of human CAIX [27 (link)], with normal rabbit serum acting as negative control. Immunohistochemical staining was performed using automated Lab Vision Autostainer 480 (ImmunoVision Technologies Co., Brisbane, CA) and polymer-based Power Vision+™ Poly-HRP IHC Kit reagents (ImmunoVision Technologies, Co.) in room temperature, as described earlier [28 (link)].
Three series of controls (primary antibody omitted, primary antibody replaced with mouse primary antibody isotype control, and staining with irrelevant CD3-antibody) were performed. Specimens of ischemic intestine were used as positive controls for HIF-1alpha staining.

Leppänen J., Helminen O., Huhta H., Kauppila J.H., Isohookana J., Haapasaari K.M., Parkkila S., Saarnio J., Lehenkari P.P, & Karttunen T.J. (2018). Weak HIF-1alpha expression indicates poor prognosis in resectable pancreatic ductal adenocarcinoma. World Journal of Surgical Oncology, 16, 127.

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Immunohistochemical Analysis of Lung Tissue

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Formalin-fixed and paraffin-embedded lung tissue specimens were cut into 3.5-µm thick sections, which were de-paraffinized in xylene and rehydrated in decreasing concentrations of ethanol. Following heat-induced or enzymatic antigen retrieval, sections were stained by using Dako EnVision Flex Kit (Dako, Glostrup, Denmark) with diaminobenzidine (DAB⁺) chromogen. Antibodies are listed in Table S1. Sections were counterstained with Mayer’s hematoxylin (Sigma-Aldrich, Steinheim, Germany). For negative controls, primary antibodies were replaced with a rabbit isotype control (Invitrogen, Carlsbad, USA). The expression of NHLRC2 was compared to the expression of collagen α1(IV) chain on the basis of the results of our previous study on the microarray analysis of lung stromal cells (12 (link)), and similarly to our previous study on IPF (9 (link)). Cluster of differentiation 68 (CD68), and alpha-smooth muscle actin (α-SMA) antibodies were used to identify macrophages and myofibroblasts, respectively.
Whole slide images were acquired at 40× magnification with a NanoZoom S60 scanner (Hamamatsu, Hamamatsu city, Japan) by Transgenic and Tissue Phenotyping core facility, Biocenter Oulu, University of Oulu.

Kreus M.A., Lehtonen S.T., Mäkinen J.M., Lappi-Blanco H.E., Laitakari K.E., Johnson S.A., Hinttala R.M, & Kaarteenaho R.L. (2023). High NHLRC2 expression is associated with shortened survival in lung adenocarcinoma. Translational Lung Cancer Research, 12(6), 1221-1235.

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Immunohistochemical Analysis of Nerve Grafts

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Grafts were fixed in 10% v/v neutral buffer formalin solution (Sigma-Aldrich, Darmstadt, Germany) paraffin embedded and sectioned. Then, the slides were deparafinnized, rehydrated and blocked. Dako Envision Flex kit was used for the immunohistochemistry assay according to manufacturer’s instructions (Dako, Agilent, Glostrup, Denmark). Briefly, nerve graft sections were incubated at 4 °C over night with rabbit anti-neurofilament 200 (nf 200) antibody (1:80, Sigma, St. Louis, MO, USA) to identify axons and S100 antibody (1:100, Sigma, St. Louis, MO, USA) to identify Schwann cells. Briefly, washes were performed, and addition of horseradish peroxidase (HRP) conjugated with goat secondary antibody against rabbit and mouse was performed. The slides were incubated at Room Temperature (RT) for 45 min. Finally, 3′3 diaminobenzidine (DAB) was added to the slides. Slides were visualized by light microscopy and images were acquired with ΙC Capture 2.2 software and processed with imageJ software version 1.52g.

Gontika I., Katsimpoulas M., Antoniou E., Kostakis A., Stavropoulos-Giokas C, & Michalopoulos E. (2018). Decellularized Human Umbilical Artery Used as Nerve Conduit. Bioengineering, 5(4), 100.

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Immunohistochemical Analysis of Cell Proliferation

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Tissues were fixed in formalin and embedded in paraffin, then sections (4 µm) were cut. Following deparaffinization with xylene, the sections were rehydrated with graded ethanol and washed with PBS, then stained with hematoxylin and eosin for histological analysis. The immunohistochemistry staining of sections was performed by using the Dako EnVision FLEX kit (Dako, Glostrup, Denmark) according to the manufacturer’s instructions as previously described [44 (link)]. Briefly, the sections were subjected to antigen retrieval in Target Retrieval Solution (Dako, Glostrup, Denmark). Endogenous peroxidase was blocked with Peroxidase-Blocking Reagent (Dako, Glostrup, Denmark). Then, the sections were incubated with primary antibody at 4 °C overnight and followed by incubation with secondary antibody FLEX/HRP (Dako, Glostrup, Denmark) at room temperatures for 30 min. Staining was developed by diaminobenzidine (DAB) substrate (Dako, Glostrup, Denmark). The stained sections were scanned by Pannoramic MIDI (3D HISTECH, Budapest, Hungary). Primary antibodies used in this study were Ki67 (Abcam, Cat# ab16667), PCNA (CST, Danvers, MA, USA, Cat# 13110), Cyclin D1 (CST, Danvers, MA, USA, Cat# 55506), E-Cadherin (CST, Danvers, MA, USA, Cat# 3195), and N-Cadherin (CST, Danvers, MA, USA, Cat# 13116).

Liu M., Lin C., Huang Q., Jia J., Guo J, & Jia R. (2023). SRSF3-Mediated Ki67 Exon 7-Inclusion Promotes Head and Neck Squamous Cell Carcinoma Progression via Repressing AKR1C2. International Journal of Molecular Sciences, 24(4), 3872.

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Immunostaining Protocol for Ki67 and p53

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Samples were immunostained for Ki67 (Dako monoclonalmouse Clone MIB‐1; Dako, Glostrup, Denmark) and p53 (Dako monoclonalmouse Clone DO‐7; Dako) using an automated immunostainer (Dako Autostainer Link; Dako) by the standard streptavidin‐biotin‐peroxidase method (Dako EnVision FLEX kit; Dako, Glostrup, Denmark). The antigen‐retrieval technique included high‐temperature heating in a microwave oven for 10 and 5 min for Ki67 and p53, respectively.

Yoshida T., Terabe T., Nagai H., Uchida F., Hasegawa S., Nagao T., Miyabe S., Ishibashi‐Kanno N., Yamagata K., Warabi E., Gosho M., Yanagawa T, & Bukawa H. (2019). Association between p62 expression and clinicopathological characteristics in oral leukoplakia. Clinical and Experimental Dental Research, 5(4), 389-397.

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Standardized H&E and IHC Staining

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H&E staining was performed on formalin fixed paraffin embedded sections using standard laboratory procedures. Immunohistochemistry was performed on paraffin sections by using the DAKO-EnVision FLEX-kit (Dako, Glostrup, Denmark). Staining was performed on an immunostainer (Autostainer; Dako, Glostrup, Denmark) according to the manufacturer’s instructions.

Krishnan A., Berthelet J., Renaud E., Rosigkeit S., Distler U., Stawiski E., Wang J., Modrusan Z., Fiedler M., Bienz M., Tenzer S., Schad A., Roth W., Thiede B., Seshagiri S., Musholt T.J, & Rajalingam K. (2020). Proteogenomics analysis unveils a TFG-RET gene fusion and druggable targets in papillary thyroid carcinomas. Nature Communications, 11, 2056.

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Immunohistochemistry Analysis of Cervical PD-1

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Immunohistochemistry (IHC) analysis for cervical biopsies was manually performed using the DAKO EnVision ™ FLEX kit (Agilent-DAKO, Santa Clara, CA). For antigenic recovery, tissue was pretreated with citrate buffer, pH 6.1 (Agilent-DAKO), and heated for 30 minutes. After blocking with endogenous peroxidase, the tissue sample was incubated with the primary monoclonal anti-PD-1 mouse antibody (ABCAM, Cambridge, UK) diluted (1:100) with DAKO antibody diluent for 1h. After washing, the sections were incubated with secondary antibody for 20 minutes and then visualized with the DAB reagent (3,3’-diaminobenzidine tetrahydrochloride, DAKO). After labeling, tissue sections were counterstained with Harris’ hematoxylin and assembled with Entellan^® (MERCK).
The IHC slides of the cervical lesion and the adjacent uninjured area were independently analyzed by two specialist pathologists. Cell areas showing brown staining were considered to be positive for the expression of PD-1 and quantified in three fields showing the highest labeling per slide, using the Gimp 2.10.18 software (GNU Image Manipulation Program, UNIX platforms, www.gimp.org). To minimize possible reading errors, we measured pixels in areas with an intense and less intense stained area in the same picture; and combined both readings to generate the final PD-1 expression value.

da Silva M.C., Medeiros F.S., da Silva N.C., Paiva L.A., Gomes F.O., Costa e Silva M., Gomes T.T., Peixoto C.A., Rygaard M.C., Menezes M.L., Welkovic S., Donadi E.A, & Lucena-Silva N. (2021). Increased PD-1 Level in Severe Cervical Injury Is Associated With the Rare Programmed Cell Death 1 (PDCD1) rs36084323 A Allele in a Dominant Model. Frontiers in Cellular and Infection Microbiology, 11, 587932.

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Immunohistochemistry of PUM Spheroids

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PUM spheroids were removed from the ULA plates using a cut 200-µl pipette tip at various time points and fixed in 10% neutral buffered formalin for 15 minutes. Spheroids were then suspended in 2% agar before being processed using the Bayer Tissue-Tek VIP E300 tissue processor (Bayer AG, Leverkusen, Germany). Processed spheroids were subsequently embedded in paraffin blocks and sectioned at 4 µm onto X-tra adhesive slides (Leica Biosystems, Wetzlar, Germany), for immunohistochemical (IHC) staining.
IHC staining was performed as previously described¹⁸ (link) using the Dako Pre-Treatment Module and the Dako Envision FLEX kit (Agilent Technologies, Santa Clara, CA), according to the manufacturer's instructions. Details of antibodies, antigen retrieval, and concentrations are provided in Table 1. Positive staining was visualized with an AEC substrate kit (Vector Laboratories, Burlingame, CA) according to the manufacturer's instructions. Sections were counterstained with Mayer's hematoxylin (VWR, Leighton Buzzard, UK), dyed blue with Scott's tap water (Leica), and mounted using Aquatex aqueous mounting medium (Sigma-Aldrich). Slides were scanned using the Leica Aperio CS2 slide scanner at 20× magnification.

Aughton K., Shahidipour H., Djirackor L., Coupland S.E, & Kalirai H. (2020). Characterization of Uveal Melanoma Cell Lines and Primary Tumor Samples in 3D Culture. Translational Vision Science & Technology, 9(7), 39.

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Immunohistochemical Analysis of Tumor Samples

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FFPE pUM and mUM samples were sectioned at 4 μm thickness and underwent antigen retrieval using the Dako pretreatment module (Agilent Technologies UK Ltd, Stockport, UK); slides were then incubated in a high‐pH bath containing Tris/EDTA buffer, pH 9.0 (Dako EnVision™ FLEX, Agilent) at 96°C for 20 min. IHC was performed using a Dako Autostainer PLUS machine, using the Dako EnVision™ FLEX Kit (Agilent) according to the manufacturer's instructions. Slides were incubated with the following antibodies for 30 min: BAP1 (cat. No sc‐28 383/C‐4, dilution 1:200; Santa Cruz Biotechnology, Dallas, TX, USA), CD3 (cat. No IR503/polyclonal, ready to use; Dako Cytomation, CA, USA), CD4 (cat. No NCL‐L‐CD4/368, dilution 1:20; Leica Biosystems, Lincolnshire, IL, USA), CD8 (cat. No M7103/ C8/144B, dilution 1:200; Dako), CD163 (NCL‐L‐CD163/10D6, dilution 1:400; Leica Biosystems), and CD38 (NCL‐L‐CD38‐290/SPC32, dilution 1:100; Leica Biosystems).
The sections were counterstained with haematoxylin. Additional sections were treated with isotype controls at the same concentration as the primary antibodies.

Figueiredo C.R., Kalirai H., Sacco J.J., Azevedo R.A., Duckworth A., Slupsky J.R., Coulson J.M, & Coupland S.E. (2020). Loss of BAP1 expression is associated with an immunosuppressive microenvironment in uveal melanoma, with implications for immunotherapy development. The Journal of Pathology, 250(4), 420-439.

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