Dako envision flex

Manufactured by Agilent Technologies

Sourced in Denmark

The Dako EnVision Flex is a laboratory equipment product manufactured by Agilent Technologies. It is designed for immunohistochemistry and in situ hybridization applications in clinical and research settings.

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Dako EnVision™ FLEX /HRP Dako Envision Flex Kit Dako EnVision FLEX detection system DAKO EnVision™ FLEX target retrieval solution high pH Dako Envision Flex Plus Mouse Link Kit Dako EnVision Flex + System Dako EnVision™ FLEX High pH Detection Kit Dako EnVision FLEX staining system Dako EnVision™ Flex Target Retrieval Solution pH 9.0 DAKO EnVision™ FLEX DAB + Substrate Chromogen System

10 protocols using dako envision flex

Immunohistochemical Analysis of CD45 in Tumor Tissue

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Representative areas for the amount of inflammation in each tumor were chosen. The immunostaining was done on 2.5 μm thick FFPE tumor tissue using a Dako Autostainer instrument (Dako, Agilent Technologies, Santa Clara, California, USA, model Link 48). Epitope retrieval was performed with Dako Flex HpH according to the manufacturer's protocol (Dako EnVision FLEX, Cat-no: K8000). Tissue sections were incubated for 20 min with primary anti-CD45 monoclonal antibodies (clones 2B11 and PD7/26, diluted 1:300; Dako, Cat-no: M0701). The secondary detection was performed with Dako EnVision TM Flex (Dako, Cat-no: K8000) for 20 min, followed by diaminobenzidine (DAB) staining for 10 min (Dako EnVision FLEX, Cat-no. K800021-2). The slides were thereafter treated with 0.5% CuSO₄ for 5 min before counterstaining with hematoxylin (Merck, Cat-no: 1.15938.0100) to visualize cell nuclei. Tissue sections were examined with a Nikon Eclipse model N i-U microscope (Nikon, Tokyo, Japan) equipped with Nikon Plan-Fluor objective lenses (2×, 10×, 20×, 40×, and 60×) and images were taken with an Infinity 2 digital camera (Lumenera Corporation, Nepean, Ontario, Canada).

Stankovic B., Bjørhovde H.A., Skarshaug R., Aamodt H., Frafjord A., Müller E., Hammarström C., Beraki K., Bækkevold E.S., Woldbæk P.R., Helland Å., Brustugun O.T., Øynebråten I, & Corthay A. (2019). Immune Cell Composition in Human Non-small Cell Lung Cancer. Frontiers in Immunology, 9, 3101.

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Immunohistochemical Analysis of CD3 and CD20

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For light microscopy, 4 μm thick sections from formalin-fixed paraffin-embedded tissue were automatically stained with hematoxylin and eosin in a Sakura Tissue-Tek Prisma instrument (Sakura Finetek, Torrance, California, USA). The immunostainings were done on a Dako Autostainer instrument (Dako, Agilent Technologies, Santa Clara, California, USA, model Link 48), and the incubation time for the primary antibodies was 20 min. CD3 was immunostained by clone SP7 (a monoclonal rabbit antibody, diluted 1:150; Thermo Scientific, Waltham, Massachusetts, USA; cat. no. RM-9107), and CD20 was immunostained by clone L26 (a mouse IgG2a antibody, diluted 1:600; Dako, cat. no. M0755). The secondary detection was performed with Dako EnVision
^TM Flex (Dako, cat. no. K8000) for 20 min, followed by diaminobenzidine (DAB) staining for 10 min. The slides were thereafter treated with CuSO4 for 5 min before contrastaining with hematoxylin. Samples were examined with a Nikon Eclipse model N
i-U microscope (Nikon, Tokyo, Japan) equipped with Nikon Plan-Fluor objective lenses (2×, 20×, and 40×) and images were taken with an Infinity 2 digital camera (Lumenera Corporation, Nepean, Ontario, Canada).

Joly-Battaglini A., Hammarström C., Stankovic B., Aamodt H., Stjärne J., Brustugun O.T., Helland Å., Øynebråten I, & Corthay A. (2016). Rituximab efficiently depletes B cells in lung tumors and normal lung tissue. F1000Research, 5, 38.

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Immunocytochemistry and Immunohistochemistry of MPC1/MPC2

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Cytoblocks were prepared for ICC and DakoEnVision™ Flex+ (K8012; Dako, Glostrup, Denmark) was applied for ICC and IHC staining as previously described [43 (link)]. After deparaffinization, unmasking epitopes and blocking of peroxidase, the slides were then incubated with the following reagents: rabbit monoclonal antibody against human MPC1(HPA045119, Sigma-Aldrich, St. Louis, MO, USA, diluted 1:500) and MPC2(ab111380, abcam, Cambridge, UK, diluted 1:500) at 4°C overnight. Negative controls were produced using the same concentration of non-immune rabbit IgG instead of the rabbit antibody to human MPC1 and MPC2.

Li Y., Li X., Kan Q., Zhang M., Li X., Xu R., Wang J., Yu D., Goscinski M.A., Wen J.G., Nesland J.M, & Suo Z. (2016). Mitochondrial pyruvate carrier function is negatively linked to Warburg phenotype in vitro and malignant features in esophageal squamous cell carcinomas. Oncotarget, 8(1), 1058-1073.

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Immunohistochemical Characterization of Neurodegeneration

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The IHC stainings were carried out automatically or manually. The automated stainings were performed on the Dako Autostainer Plus (DakoCytomation, Glostrup, Denmark) using the Dako EnVision Flex detection system (DakoCytomation, Glostrup, Denmark), according to the manufacturer’s instructions. The Bright Vision detection system (IL Immunologic, Duiven, The Netherlands), with a Romulin AEC for antigen detection (BioCare Medical, Pacheco, CA, U.S.A.), was used for the manual stainings. The antibodies (Ab) used and the pretreatment strategies applied are listed in Table 1.Table 1

Immunohistochemical stains

Antibody	Clone	Company/Code	Dilution	Pretreatment
Amyloid β (Aβ)	6F/3D	Dako-Agilent/M0872	1:50	98–100% FA
Embryonic lethal abnormal visual system proteins (nELAV) 3 and 4 human homolog HuC/HuD (HuC/HuD)	16A11	ThermoFisher Scientific/A-21271	1:2000	ac, CB
NEUronal Nuclei (NeuN)	A60	Milipore/MAB377	1:2000	CB
Hyperphosphorylated (Ser202/Thr205) τ (TAU8)	PHF-TAU-AT8	Fisher Sientific-Invitrogen/MN1020	1:1000
Synaptophysin (SYP)	SY38	Dako-Cytomation/M0776	1:50	CB

Dako Autostainer Plus (Dako Cytomation) was used for Aβ, Tau8 and SYP, while the HuC/HuD and NeuN were carried out manually, incubation at room temperature for 1 h. Autoclave (ac), formic acid (FA), citrate buffer pH 6.0 (CB)

Libard S, & Alafuzoff I. (2019). Alzheimer’s disease neuropathological change and loss of matrix/neuropil in patients with idiopathic Normal Pressure Hydrocephalus, a model of Alzheimer’s disease. Acta Neuropathologica Communications, 7, 3.

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Histological Diagnosis and Immunohistochemical Profiling of Adrenocortical Carcinoma

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Histologic diagnosis of ACC was performed by reference pathologist on tumor tissue removed at surgery. Tumor specimens were evaluated according to the Weiss System where the presence of three or more criteria highly correlates with malignant behavior [34 ].
Ki67 index was evaluated as proliferation marker to assess ACC prognosis using anti-human Ki67 monoclonal MIB1 antibody (Dako, Carpenteria, CA, US). Ki67 positive nuclei were counted on 1.000 tumor cells and Ki67 was expressed as the percentage of proliferating cells.
Tumor stage was evaluated according to the revised TNM classification of ACC proposed by the European Network for the Study of Adrenal Tumors [13 (link)].
For immunohistochemical analysis, section of 3μm were deparaffinized, hydrated with grade ethanol concentrations until distilled water. Serial sections of the same specimen were immunostained with mouse monoclonal anti-ALDH6A1, anti-Fascin-1, anti-CAP-1, anti-Lamin A/C, anti-Ferredoxin-reductase, anti-Transferrin (dilution 1:50) antibodies after treatement with 3.0% hydrogen peroxidase in PBS (Dako Wash Buffer 10x). Immunohistochemical analysis was carried out using DAKO EnVision™ FLEX (Dako, Carpenteria, CA, US).. Negative control was performed with a non-immune serum.

Poli G., Ceni E., Armignacco R., Ercolino T., Canu L., Baroni G., Nesi G., Galli A., Mannelli M, & Luconi M. (2015). 2D-DIGE proteomic analysis identifies new potential therapeutic targets for adrenocortical carcinoma. Oncotarget, 6(8), 5695-5706.

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Immunohistochemical Analysis of Myb Expression

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Immunohistochemical staining for Myb was performed using the Dako EnVision+ System (Dako, Glostrup, Denmark). Briefly, 4um TMA sections were deparaffinized with xylene and ethanol. Heat-induced epitope retrieval was completed with EnVision Module at 97 °C for 20 min in pH 9.0 buffer. Endogenous peroxidase activity was blocked with Dako EnVision Flex. Tissue sections were then incubated with an anti-Myb monoclonal antibody (Genetex, Irvine, CA). The reaction was visualized using the Dako EnVision Flex+ System. External positive and negative controls were performed.
Myb was scored based on both % cells staining and the intensity of staining. The % of cells showing any staining was visually assessed along with the intensity of Myb staining using a 3-point scale: (0: absent; 1: weak; 2: moderate; 3: intense). Scoring was performed by two independent board-certified pathologists (AvZ and SA) with discrepant scores re-examined by both observers to achieve a consensus score.

Park S., Vora M., van Zante A., Humtsoe J., Kim H.S., Yom S., Agarwal S, & Ha P. (2020). Clinicopathologic implications of Myb and Beta-catenin expression in adenoid cystic carcinoma. Journal of Otolaryngology - Head & Neck Surgery, 49, 48.

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Immunohistochemical Analysis of MCM3 and Ki-67

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IHC was performed on tissue microarray sections of 4 μm thickness using a DAKO Omnis platform (Agilent Technologies, Santa Clara, CA). Heat-induced epitope retrieval was performed on board, followed by antibody incubation in Dako EnVision FLEX (Agilent Technologies, Santa Clara, CA). For MCM3, the DAKO Omnis protocol H30-X-30 was utilized with recombinant rabbit monoclonal anti-MCM3 antibody (dilution 1/1000, clone EPR7080, catalogue # ab128923; Abcam, Cambridge, UK). For Ki-67, the DAKO Omnis protocol L20-X-20 was used with mouse monoclonal anti-Ki-67 antibody (ready-to-use, clone Mib-1, catalogue # GA626; Agilent Technologies, Santa Clara, CA). Nuclear expression in tumor cells was scored in 5% increments. Based on the distribution, no naturally occurring cut-off was apparent. Therefore, scores were categorized into 3 relatively equally sized groups after several iterations to optimize for prognostic stratification (For MCM3: <40%, 40% to 75%, >75%; for Ki-67 <20%, 20% to 30%, >30%). TMA cores with less than 10% tumor content were excluded from study. A subset of cases was scored again by a second observer blinded to the initial scores. Previously generated immunohistochemical and chromogenic in situ hybridization (CISH) data for p53, p16, RB1, and CCNE1 were used for correlative analysis [5 (link), 18 (link), 21 (link)].

Kang E.Y., Millstein J., Popovic G., Meagher N.S., Bolithon A., Talhouk A., Chiu D.S., Anglesio M.S., Leung B., Tang K., Lambie N., Pavanello M., Da-anoy A., Lambrechts D., Loverix L., Olbrecht S., Bisinotto C., Garcia-Donas J., Ruiz-Llorente S., Yagüe-Fernandez M., Edwards R.P., Elishaev E., Olawaiye A., Taylor S., Ataseven B., du Bois A., Harter P., Lester J., Høgdall C.K., Armasu S.M., Huang Y., Vierkant R.A., Wang C., Winham S.J., Heublein S., Kommoss F.K., Cramer D.W., Sasamoto N., van-Wagensveld L., Lycke M., Mateoiu C., Joseph J., Pike M.C., Odunsi K., Tseng C.C., Pearce C.L., Bilic S., Conrads T.P., Hartmann A., Hein A., Jones M.E., Leung Y., Beckmann M.W., Ruebner M., Schoemaker M.J., Terry K.L., El-Bahrawy M.A., Coulson P., Etter J.L., LaVigne-Mager K., Andress J., Grube M., Fischer A., Neudeck N., Robertson G., Farrell R., Barlow E., Quinn C., Hettiaratchi A., Casablanca Y., Erber R., Stewart C.J., Tan A., Yu Y., Boros J., Brand A.H., Harnett P.R., Kennedy C.J., Nevins N., Morgan T., Fasching P.A., Vergote I., Swerdlow A.J., dos Reis F.J., Maxwell G.L., Neuhausen S.L., Barquin-Garcia A., Modugno F., Moysich K.B., Crowe P.J., Hirasawa A., Heitz F., Karlan B.Y., Goode E.L., Sinn P., Horlings H.M., Høgdall E., Sundfeldt K., Kommoss S., Staebler A., Wu A.H., Cohen P.A., DeFazio A., Lee C.H., Steed H., Le N.D., Gayther S.A., Lawrenson K., Pharoah P.D., Konecny G., Cook L.S., Ramus S.J., Kelemen L.E, & Köbel M. (2021). MCM3 is a novel proliferation marker associated with longer survival for patients with tubo-ovarian high-grade serous carcinoma. Virchows Archiv : an international journal of pathology, 480(4), 855-871.

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Immunohistochemical Detection of PD-L1 in Tissue Samples

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Slides were stained for routine diagnostics, over a period of several years. Formalin‐fixed paraffin‐embedded blocks were cut into 3‐µm sections with a Leica RM2255 Automated Microtome (Leica Biosystems B.V., Amsterdam, the Netherlands). Sections were placed on microscope slides and dried at either 60°C for 30 min to 16 h, or at 37°C for 72 h. After being dried, the slides were deparaffinised, and antigen retrieval was performed in citrate buffer (Target Retrieval Solution, pH 6) for 40 min. Immunohistochemistry (IHC) was performed according to a laboratory‐developed test protocol. Slides were stained with the Dako Omnis immunostainer and Dako EnVision Flex+ reagents and 1:20 dilution of PD‐L1 clone 22C3 (Dako Omnis, Dako Agilent Technologies, Leuven, Belgium). The IHC slides were then counterstained with haematoxylin, and coverslips were applied. Tonsil and placental tissue were used as positive controls for PD‐L1 expression.

Hondelink L.M., Hüyük M., Postmus P.E., Smit V.T., Blom S., von der Thüsen J.H, & Cohen D. (2021). Development and validation of a supervised deep learning algorithm for automated whole‐slide programmed death‐ligand 1 tumour proportion score assessment in non‐small cell lung cancer. Histopathology, 80(4), 635-647.

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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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Elastin Expression in Human Lungs

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Elastin immunohistochemistry was performed to evaluate the expression of elastin in human lung tissues. Briefly, mouse anti-elastin antibody (ab77804, Abcam) was incubated with human lung tissues for 60 min after blocking. Tissues were washed and subjected to DAKO Envision FLEX (Agilent Technologies, Inc. Santa Clara, CA). After the tissues were washed, DAB was used as the chromogen. Then, the tissues were washed again and stained by Mayer's hemalum solution.

Tokita S., Sugiyama K., Wakayama T., Arifuku H., Otsuji N., Sugitate K., Owada T., Koyama K., Hirata H., Arima M., Ueda Y, & Fukushima Y. (2021). Elevation of anti-elastin antibody in patients with asthma. Asian Pacific journal of allergy and immunology.

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