Envision doublestain system

Manufactured by Agilent Technologies

Sourced in Denmark, Germany

The EnVision Doublestain system is a laboratory equipment product from Agilent Technologies. It is designed to enable the simultaneous detection and visualization of two target molecules or antigens within a single tissue sample.

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Lab products found in correlation

3 3 diaminobenzidine tetrahydrochloride hydrate, by Merck Group (3 mentions) Diethylnitrosamine, by Merck Group (3 mentions) Apoptag peroxidase in situ kit, by Merck Group (3 mentions) Vectastrain abc kit, by Vector Laboratories (2 mentions) Methylene blue, by Merck Group (2 mentions) Anti pcna antibody, by BioLegend (2 mentions) Mouse monoclonal proliferating cell nuclear antigen antibody, by BioLegend (2 mentions) Avidin biotin horseradish peroxidase complex abc kit, by Vector Laboratories (2 mentions) Apoptag peroxidase in situ apoptosis detection kit, by Merck Group (1 mentions) Protease xxiv, by Merck Group (1 mentions) Hpa003157, by Atlas Antibodies (1 mentions) Diethylnitrosamine den, by Merck Group (1 mentions) Vectastain abc kit, by Vector Laboratories (1 mentions) Opal 4 color fluorescent ihc kit, by PerkinElmer (1 mentions) Axio observer, by TissueGnostics (1 mentions) Tissuefax, by TissueGnostics (1 mentions) Tissuequest, by TissueGnostics (1 mentions) 4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions) Collagenase type 4, by Thermo Fisher Scientific (1 mentions) Anti cd3 polyclonal, by Agilent Technologies (1 mentions)

16 protocols using envision doublestain system

Evaluating Cell Proliferation and Apoptosis

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To evaluate the effect of PRBE and PCA on cell proliferation and apoptosis, double-staining procedures were carried out using an EnVision Doublestain system from Dako (Hamburg, Germany). Liver sections were stained with anti-PCNA antibody (Biolegend, USA) at 1 : 2000 dilution and anti-GST-P antibody (MBL, Japan) at 1 : 1000 dilution, following the manufacturer’s instructions. The number of PCNA-positive cells was counted under a light microscope as described in our previous study⁸.
The terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was used to identify apoptotic hepatocytes. A double-labeling assay for TUNEL and GST-P was carried out using an ApopTag Peroxidase in situ kit (Merck, Germany) and EnVision Doublestain system (Dako, Denmark) according to Thumvijit et al. (2014)^{15 (link)}. The number of apoptotic labeled cells was recorded as in our previous reports⁸.

Punvittayagul C., Luangsuphabool T, & Wongpoomchai R. (2022). Protocatechuic acid as a potent anticarcinogenic compound in purple rice bran against diethylnitrosamine-initiated rat hepatocarcinogenesis. Scientific Reports, 12, 10548.

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Quantifying Islet Vasculature Density

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The consecutive sections were stained for CD31 and synaptophysin. Primary antibodies (S1 Table) were added and thereafter visualized using Dako EnVision Doublestain system (DAB+/Permanent Red). Sections were counterstained with hematoxylin (Histolab) and photographed using a Zeiss Palm Microbeam Ⅳ microscope at 20× magnification. The CD31 positive regions within islets was evaluated with assistance of ImageJ software. Ten islets per donor was analyzed; the islet area (μm²) and the length of all CD31 positive regions (μm) found within these islets were noted. The total length of CD31 positive regions per total islet area (vascular density) was calculated.

Granlund L., Hedin A., Korsgren O., Skog O, & Lundberg M. (2022). Altered microvasculature in pancreatic islets from subjects with type 1 diabetes. PLOS ONE, 17(10), e0276942.

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Hepatocellular Carcinoma Induction Protocol

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Diethylnitrosamine, 3,3′-diaminobenzidine tetrahydrochloride hydrate and VA were purchased from Sigma-Aldrich (St. Louis, MO, USA). 1,2-Dimethylhydrazine dihydrochloride was obtained from TCI (Tokyo, Japan). Methylene blue and an ApopTag^® Peroxidase In Situ Apoptosis Detection Kit were purchased from Merck (Darmstadt, Germany). Anti-rat glutathione S-transferase placental form was obtained from MBL (Nagoya, Japan). A Vectastrain ABC kit was obtained from Vector Laboratories, Inc. (Burlingame, CA, USA). An EnVision Doublestain system was purchased from Dako (Hamburg, Germany). All other chemicals were of analytical grade.

Punvittayagul C., Chariyakornkul A., Jarukamjorn K, & Wongpoomchai R. (2021). Protective Role of Vanillic Acid against Diethylnitrosamine- and 1,2-Dimethylhydrazine-Induced Hepatocarcinogenesis in Rats. Molecules, 26(9), 2718.

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Dual IHC Visualization of Collagen IV and Biglycan

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An immunohistochemistry (IHC) double-staining procedure visualised col IV and biglycan on 4 µm sections from formalin-fixed, paraffin-embedded biopsies using the EnVision Doublestain System (K5361, Dako, Glostrup, Denmark) according to the manufacturer's instructions. Single-staining tests for each antibody showed that the locations for the two proteins were not overlapping, thereby enabling quantification of each target in the double staining procedure. Antibodies were assessed for specificity and optimal antigen retrieval methods.21 (link) Antigen retrieval was performed with 0.2% bovine testicular hyaluronidase (Sigma type I-S, buffer 20mM sodium acetate, 150mM sodium chloride, pH 5.2) for 30 min and subsequently with 20 µg/mL protease XXIV in PBS (Subtilisin, Carlsberg, Sigma) for 20 min (both room temperature). Primary antibodies against col IV α1 chain (Abcam 6586, concentration 1 mg/mL, used at 1:4000) and biglycan (Atlas antibodies, Uppsala, Sweden, HPA003157, concentration 0.1 mg/mL, used at 1:1750) were incubated for 1 hour at room temperature. Secondary antibodies produced brown (col IV) or red (biglycan) precipitates. Sections were counterstained with Mayer's haematoxylin for visualisation of nuclei.

Müller C., Andersson-Sjöland A., Schultz H.H., Eriksson L.T., Andersen C.B., Iversen M, & Westergren-Thorsson G. (2017). Early extracellular matrix changes are associated with later development of bronchiolitis obliterans syndrome after lung transplantation. BMJ Open Respiratory Research, 4(1), e000177.

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Hepatic Carcinogenesis Induction Protocol

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Diethylnitrosamine (DEN) and 3, 3’-diaminobenzidine tetrahydrochloride hydrate were obtained from Sigma-Aldrich (St. Louis, MO, USA). ApopTag Peroxidase in situ kit was purchased from Merck (Darmstadt, Germany). Rabbit polyclonal antibody to rat glutathione S-transferase placental form (GST-P) was purchased from MBL (Nagoya, Japan). Protocatechuic acid (PCA) was purchased from Alfa-Aesar (Karlsruhe, Germany). Vectastain ABC kit was purchased from Vector Laboratories, Inc. (Burlingame, CA, USA). EnVision Doublestain system was purchased from Dako (Glostrup, Denmark). All other chemicals were analytical grade.

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Quantitative Analysis of CD8+ T Cells

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Localization of CD8⁺ T subsets was assessed as described.^{4 (link)} Briefly, slides were prepared from 4-µm sections of paraffin-embedded tissue blocks and immune stained using in-house optimized protocols. For each LN, serial sections were stained singly with antibodies against BCL6, Gag-p24, CD8, and a DAB visualization kit (Envision Double Stain system; Dako) for bright-field microscopy. Alternatively, we used the Opal 4-Color Fluorescent IHC Kit (PerkinElmer) for immunofluorescence microscopy light. Slides were mounted and viewed using an Axio Observer and TissueFAXS imaging software (TissueGnostics, Vienna, Austria). Quantitative imaging analysis was conducted with TissueQuest (TissueGnostics). Medians of cell density in the GCs were used to perform statistical analyses.

Ogunshola F.J., Smidt W., Naidoo A.F., Nkosi T., Ngubane T., Khaba T., Baiyegunhi O.O., Mahlobo B., Rasehlo S., Ngema N., Jajbhay I., Dong K.L., Ramsuran V., Pansegrouw J., Ndung’u T., Walker B.D., de Oliveria T, & Ndhlovu Z.M. (2022). Hypermethylation at the CXCR5 gene locus limits trafficking potential of CD8+ T cells into B-cell follicles during HIV-1 infection. Blood Advances, 6(6), 1904-1916.

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Carcinogenesis Induction and Histological Analysis

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Diethylnitrosamine and 3,3′-diaminobenzidine tetrahydrochloride hydrate were purchased from Sigma Aldrich (St. Louis, MO, USA). Collagenase type IV and 4′,6-diamidino-2-phenylindole (DAPI) were obtained from Invitrogen (Waltham, MA, USA). The 1,2-dimethylhydrazine dihydrochloride was purchased from TCI (Tokyo, Japan). The ApopTag Peroxidase in situ kit and methylene blue were obtained from Merck (Darmstadt, Germany). The Vectastrain ABC kit was obtained from Vector Laboratories, Inc. (Burlingame, CA, USA). The EnVision Doublestain system was provided by Dako (Glostrup, Denmark). All other chemicals were analytical grade.

Punvittayagul C., Chariyakornkul A., Sankam P, & Wongpoomchai R. (2021). Inhibitory Effect of Thai Purple Rice Husk Extract on Chemically Induced Carcinogenesis in Rats. Molecules, 26(2), 360.

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Immunocytochemical Characterization of Single Cells

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Cytospins of SCS were fixed with methanol. The DakoCytomation EnVision Doublestain system was used for chromogenic immunocytochemistry. For fluorescent immunocytochemistry, cytospins were blocked with Image-iT FX, incubated with primary antibodies (anti-CD3 [polyclonal], Dako; anti-CD1a [O10], Abcam), washed, labeled with secondary antibodies (Alexa Fluor 488 and Alexa Fluor 596, Invitrogen), washed and mounted with ProLong Gold antifade reagent with DAPI (4,6 diamidino-2-phenylindole) (Invitrogen Molecular Probes). Isotype-matched antibodies were used as negative controls.

West J.A., Olsen S.L., Mitchell J.M., Priddle R.E., Luke J.M., Åkefeldt S.O., Henter J.I., Turville C, & Kannourakis G. (2014). Polyclonal T-Cells Express CD1a in Langerhans Cell Histiocytosis (LCH) Lesions. PLoS ONE, 9(10), e109586.

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Quantification of Fibroblast Activation and Proliferation

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For quantification of activation and proliferation of fibroblasts, double immunohistological staining of alpha smooth muscle actin (αSMA) and proliferating cell nuclear antigen (PCNA) was performed. Briefly, histological sections were incubated with primary antibodies raised against αSMA (Sigma, USA; 1:100) and PCNA (Dako, USA; 1:100) revealed, respectively, by Fast red and Diaminobezidine according to the manufacturer’s instructions (EnVision Doublestain System, Dako, USA). Morphometric analysis was performed for the quantification of total fibroblasts and proliferating fibroblasts using an image analysis Image-Pro Plus system (Media Cybernetics, USA). Results were expressed as number of cells per μm² of injured tissue.

Cogliati B., Vinken M., Silva T.C., Araújo C.M., Aloia T.P., Chaible L.M., Mori C.M, & Dagli M.L. (2015). Connexin 43 deficiency accelerates skin wound healing and extracellular matrix remodeling in mice. Journal of dermatological science, 79(1), 50-56.

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Immunohistochemical Analysis of Liver Apoptosis

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The double-staining procedures were performed by using an EnVision Doublestain system from Dako (Glostrup, Denmark). Liver sections were performed for immunohistochemical staining with anti-PCNA antibody (Biolegend, San Diego, CA, USA) and anti-rat GST-P antibody according to manufacturer’s instructions. The number of PCNA-positive cells was counted both inside and in the area surrounding GST-P positive foci. The PCNA labeling index (%) was determined by counting at least 2000 hepatocytes.
Apoptotic hepatocytes were determined by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. The double immunostaining for TUNEL and GST-P was performed by using an ApopTag Peroxidase in situ kit and an EnVision Doublestain system according to Thumvijit et al. (2014) [34 ]. The number of apoptotic cells was counted both inside and surrounding the areas of GST-P-positive foci. The apoptotic labeling index (%) was determined by counting at least 2000 hepatocytes.

Punvittayagul C., Chariyakornkul A., Sankam P, & Wongpoomchai R. (2021). Inhibitory Effect of Thai Purple Rice Husk Extract on Chemically Induced Carcinogenesis in Rats. Molecules, 26(2), 360.

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