Envision flex dab

Manufactured by Agilent Technologies

Sourced in Denmark

The Envision Flex DAB is a laboratory equipment product offered by Agilent Technologies. It is designed for chromogenic detection in immunohistochemistry (IHC) and in situ hybridization (ISH) applications. The product provides a stable, sensitive, and specific detection system for visualizing target antigens or nucleic acid sequences in tissue samples.

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DAKO EnVision™ FLEX DAB + Substrate Chromogen System EnVision FLEX DAB + Chromogen EnVision™ FLEX Substrate buffer + DAB + Chromogen EnVision™ FLEX DAB+ Substrate Chromogen System Envision Flex Dab kit EnVision FLEX

8 protocols using envision flex dab

Histopathological Prion Protein Analysis

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Histopathology and PrP immunohistochemistry were performed as previously described [28 (link)]. Briefly, deparaffinized and rehydrated sections were immersed in Tris-buffered saline-Tween 20 (TBS-T) and endogenous peroxidase blocked by Envision Flex Peroxidase Blocking Reagent (Dako) for 10 minutes. Sections were washed, immersed in 1.5 mmol/L hydrochloric acid, microwaved for 15 minutes and incubated with 3F4 (1:1000) for 1 hour. After washing and incubation with Envision Flex/HRP polymer for 30 minutes (Dako), sections were treated with Envision Flex DAB (Dako) to show the immunostaining.

Cali I., Miller C.J., Parisi J.E., Geschwind M.D., Gambetti P, & Schonberger L.B. (2015). Distinct pathological phenotypes of Creutzfeldt-Jakob disease in recipients of prion-contaminated growth hormone. Acta Neuropathologica Communications, 3, 37.

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Immunohistochemical Analysis of Prion Protein

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For the USA cases, the formalin-fixed brain was treated as previously described [18 (link)]. Tissue was deparaffinized, rehydrated, and immersed in Tween 20-Tris buffered saline. The endogenous peroxidase was blocked by the Envision Flex Peroxidase Blocking Reagent (Dako North America Inc., Carpinteria, CA, USA) for 10 min, which was followed by several washes. Sections were immersed in hydrochloric acid (1.5 mmol/L), microwaved for 15 min, and incubated with purified 3F4 Ab (1:1000) for 1 hour. Sections were washed and incubated with Envision Flex/HRP polymer for 30 min (Dako). The Envision Flex DAB (Dako) was used to show the immunostaining. Brain tissue blocks from the only UK-autopsied case were from 1985 and not suitable for modern immunocytochemistry.

Brennecke N., Cali I., Mok T.H., Speedy H., Hosszu L.L., Stehmann C., Cracco L., Puoti G., Prior T.W., Cohen M.L., Collins S.J., Mead S, & Appleby B.S. (2021). Characterization of Prion Disease Associated with a Two-Octapeptide Repeat Insertion. Viruses, 13(9), 1794.

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Immunohistochemical Analysis of CD44v6 in Tumors

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Immunohistochemistry (IHC) was performed on six A431 and UM-SCC-74B tumors, fixated in formalin directly after dissection. Tumors were paraffin-embedded, sectioned and deparaffinized. Antigen retrieval was achieved by microwaving (10+15 min) in citrate buffer (DAKO, S2369) or Tris-EDTA buffer (DAKO, S2367). Immunostaining with anti-CD44v6 (AbD Serotec) was performed according to manufactures instructions followed by detection with the EnVision FLEX system (DAKO, K8000). The reaction was visualized by EnVision FLEX DAB+ (DAKO). Mayer’s hematoxylin (DAKO) was used as counterstain. Images of the IHC staining (magnification x10) were obtained using a Nikon D3000 digital camera mounted on an inverted Nikon Diaphot-TMD 290 microscope.

Haylock A.K., Nilvebrant J., Mortensen A., Velikyan I., Nestor M, & Falk R. (2017). Generation and evaluation of antibody agents for molecular imaging of CD44v6-expressing cancers. Oncotarget, 8(39), 65152-65170.

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TATI Immunohistochemical Staining Protocol

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IHC staining of TATI was analyzed in TMA slides. Deparaffinization and rehydration of the tissue slides was performed with Sakura Tissue-Tek DRS. The HIER method (heat induced epitope retrieval) was performed with the Pretreatment Module, Agilent Dako (Dako Denmark Aps, Glostrup, Denmark) to improve antigen retrieval in the samples. Endogenous peroxidase blocking was performed with EnVision Flex peroxidase-blocking reagent (Dako). A monoclonal TATI antibody (MAb 6E8) [37 (link)] was used as the primary antibody. Dako REAL Antibody Diluent S2022 (Dako) was used for antibody dilutation. EnVision Flex/HRP SM802 DM827 (Dako) was used as a secondary antibody. The chromogen was EnVision Flex DAB (Dako). Hematoxylin was used for counterstaining. Staining was performed with Autostainer 480 (Thermo Fisher Scientific, Vantaa, Finland). After the staining procedure, the specimen was dehydrated and then mounted with Pertex Histolab mounting media (Histolab Products Oy, Gothenburg, Sweden). We have not found evidence of non-specific staining while previously applying this method for TATI IHC and the specificity of the TATI antibody is described in earlier studies [34 (link),36 (link),37 (link)].

Sjöblom A., Stenman U.H., Hagström J., Jouhi L., Haglund C., Syrjänen S., Mattila P., Mäkitie A, & Carpén T. (2021). Tumor-Associated Trypsin Inhibitor (TATI) as a Biomarker of Poor Prognosis in Oropharyngeal Squamous Cell Carcinoma Irrespective of HPV Status. Cancers, 13(11), 2811.

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Immunohistochemical Analysis of Collagen Types

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After culture, matrices were embedded in HistoGel, fixed in 4% paraformaldehyde and paraffin wax-embedded for sectioning and staining for human collagen type IV (mouse anti-human collagen IV monoclonal antibody 2150–0121, Bio-Rad, CA, USA) and human collagen type I/III (rabbit anti-human collagen I/III polyclonal antibody 2150–2210, Bio-Rad, CA, USA). After automated antigen retrieval processing (PT Link Machine, DAKO, Denmark), sections were washed three times for five minutes in wash buffer (EnVision Flex Wash Buffer, DAKO, Denmark), blocked for ten minutes in blocking reagent (EnVision FLEX peroxidase-blocking reagent, DAKO, Denmark), washed a further three times, incubated with primary antibody diluted in substrate buffer (EnVision Flex Substrate Buffer, DAKO, Denmark) for one hour at room temperature, washed three times, and incubated with secondary antibody (EnVision FLEX/HRP, DAKO, Denmark) for 30 minutes at room temperature. After three washes, 3,3-diaminobenzidine tetrahydrochloride (DAB) solution (EnVision FLEX DAB, DAKO, Denmark) was added with H&E counterstain. Acellular matrix (consisting of rat type I collagen) was used as a negative control, and normal human kidney was used as a positive control.

Waters J.P., Richards Y.C., Skepper J.N., Southwood M., Upton P.D., Morrell N.W., Pober J.S, & Bradley J.R. (2017). A 3D tri-culture system reveals that activin receptor-like kinase 5 and connective tissue growth factor drive human glomerulosclerosis. The Journal of pathology, 243(3), 390-400.

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Immunohistochemical Analysis of Cyclin D1 and MIB-1

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We collected all available tumor blocks from the study groups, based on their diagnostic slides. To prepare the tumor samples for immunohistochemical staining, we used the following protocol for both cyclin D1 and MIB-1: 4 µm thick slides were prepared from formalin-fixed paraffin-embedded (FFPE) tumor blocks, deparaffinized in xylene, and rehydrated with ethanol. Antigen retrieval was carried out by heat induced epitope retrieval (HIER), and the retrieval solution was pH 9, 15 min 98 °C. After that we deployed blocking of endogenous peroxidase and added primary antibody. For cyclin D1 we used the monoclonal rabbit anti-cyclin D1 antibody (SP4), ab 16663, Abcam with incubation time of O/N +5. For MIB-1, Dako Agilent monoclonal mouse antibody Ki-67/MIB-1 7240 (Dako Agilent, Santa Clara, USA) was used with incubation time of O/N +5. Secondary antibody (EnVision Flex, Dako, Clostrup, Denmark), chromogen (EnVision Flex DAB, Dako, Clostrup, Denmark) and substrate (Dako Mayer’s Hematoxylin, Dako, Clostrup, Denmark) were deployed. The staining process was performed with an Autostainer 480S (LabVision, UK).

Markkanen A., Aro K., Laury A.R., Mäkitie A.A., Haglund C., Atula T, & Hagström J. (2022). Increased MIB-1 expression in salivary gland pleomorphic adenoma that recurs and undergoes malignant transformation. Scientific Reports, 12, 9029.

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Immunohistochemical Staining of FFPE Tumor Sections

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Serial sections were cut from the selected FFPE tumor blocks (N = 129) and mounted on FLEX IHC Microscope Slides (K8020, DAKO, Glostrup, Denmark). The pretreatment processes were performed using PT Link (DAKO). Heat-induced epitope retrieval was achieved with Envision Target Retrieval Solution (DAKO) at pH 9 and 97 °C for 20 min.
Staining was performed using a DAKO Autostainer Link 48 (DAKO).
Endogenous peroxidase activity was blocked by Envision FLEX Peroxidase-Blocking Reagent (DAKO). The primary antibodies were mouse monoclonal anti-CD3 (code M7254, DAKO) diluted 1:600, and anti-CD8 (code M7103, DAKO) diluted 1:300. The primary antibodies were diluted with Envision Flex antibody diluent (code S2022 DAKO).
Primary antibodies were incubated for 30 min at room temperature, and for amplification Envision Flex + Mouse(Linker) (DAKO) was used for 20 min. Bound antibodies were detected using Envision FLEX/HRP (DAKO) and visualized by Envision FLEX DAB (DAKO) and chromogene diluted in Envision Flex Substrate Buffer (DAKO). To enhance the immunohistochemical stains, the sections were incubated in 0.5% CuSO₄ in TBS buffer pH 7.6 for 10 min. Meyer’s hematoxylin (Merck, Damstadt, Germany) was used as counterstain, and finally, the histological slides were coverslipped with Tissue-Tek PERTEX (Histolab Products AB, Göteborg, Sweden).

Eriksen A.C., Andersen J.B., Kristensson M., dePont Christensen R., Hansen T.F., Kjær-Frifeldt S, & Sørensen F.B. (2017). Computer-assisted stereology and automated image analysis for quantification of tumor infiltrating lymphocytes in colon cancer. Diagnostic Pathology, 12, 65.

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Immunohistochemical Staining of Cytokeratin AE1/AE3

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Serial sections were cut from the selected tumour blocks (N = 129) and mounted on FLEX IHC Microscope Slides (K8020, Agilent DAKO products, Glostrup, Denmark). The pretreatment processes were performed using PT Link (DAKO). Heat-induced epitope retrieval was achieved with Envision Target Retrieval Solution (DAKO) at pH 9 and 97 °C for 20 min. Staining was performed using a DAKO Autostainer Link 48 (DAKO). Endogenous peroxidase activity was blocked by Envision FLEX Peroxidase-Blocking Reagent (DAKO). The primary antibody was mouse monoclonal cytokeratin AE1/AE3 (code M3515, DAKO) diluted 1:250 with Envision Flex antibody diluent (code S2022 DAKO). Primary antibody was incubated for 30 min. at room temperature, and for amplification Envision Flex+ Mouse (Linker) (DAKO) was used for 20 min. Bound antibodies were detected by Envision FLEX/HRP (DAKO) and visualised by Envision FLEX DAB (DAKO) with chromogen diluted in Envision Flex Substrate Buffer (DAKO). Meyer’s hematoxylin (Merck, Darmstadt, Germany) was used as counterstain and finally, the histological slides were cover slipped with Tissue-Tek PERTEX (Histolab Products AB, Göteborg, Sweden).

Eriksen A.C., Andersen J.B., Lindebjerg J., dePont Christensen R., Hansen T.F., Kjær-Frifeldt S, & Sørensen F.B. (2018). Does heterogeneity matter in the estimation of tumour budding and tumour stroma ratio in colon cancer?. Diagnostic Pathology, 13, 20.

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