Immunohistochemical study was performed as follows. In brief, Paraffin-embedded sections from 562 patients were heated for 40 min at 98 °C by heater in Target Retrieval Solution High pH (DAKO, Carpinteria, CA). Then sections were incubated with 3% hydrogen peroxide to block endogenous peroxidase activity and the sections were immersed in protein block (DAKO protein block, serum free) to block nonspecific binding. The specimens were incubated with anti-FGFR2-IIIb (dilution, 1:333) for 30 min at room temperature, and anti-FGFR2-IIIc (dilution, 1:333) for overnight at 4 °C. These sections were incubated with mouse linker (ENVISION FLEX Mouse+, DAKO) for 20 min, and peroxidase-labeled polymer (ENVISION FLEX/HRP, DAKO) for 20 min. The sections were counterstained with hematoxylin.
Envision flex mouse
Manufactured by Agilent Technologies
Sourced in United States
The ENVISION FLEX Mouse+ is a lab equipment product from Agilent Technologies. It is a mouse-based input device designed for use with compatible software and systems. The core function of the ENVISION FLEX Mouse+ is to provide a means of user interaction and control within the intended software environment.
Automatically generated - may contain errors
Lab products found in correlation
Envision flex hrp, by Agilent Technologies (5 mentions)
Envision flex peroxidase blocking reagent, by Agilent Technologies (2 mentions)
Protein block, by Agilent Technologies (1 mentions)
Pten clone 6h2, by Agilent Technologies (1 mentions)
Ki 67 clone mib 1, by Agilent Technologies (1 mentions)
Pt link module, by Agilent Technologies (1 mentions)
Autostainer, by Agilent Technologies (1 mentions)
Ab84989, by Abcam (1 mentions)
Cbl404, by Merck Group (1 mentions)
3 3 diaminobenzidine (dab), by Agilent Technologies (1 mentions)
Avidin biotin blocking kit, by Vector Laboratories (1 mentions)
Tenascin c, by Abcam (1 mentions)
Vectastain elite abc kit, by Abcam (1 mentions)
Envision flex rabbit, by Agilent Technologies (1 mentions)
H2a x d17a3 xp, by Cell Signaling Technology (1 mentions)
Mib 1, by Agilent Technologies (1 mentions)
Mnf116, by Agilent Technologies (1 mentions)
Envision flex, by Agilent Technologies (1 mentions)
Nch 38, by Agilent Technologies (1 mentions)
7 protocols using envision flex mouse
1
Immunohistochemical Study of FGFR2 Isoforms
2
Immunohistochemical Profiling of FFPE Tissues
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Immunostaining was performed on 2 μm FFPE tissue sections. Deparaffinisation and antigen retrieval were done in a PT Link module (Dako, Denmark) and immunohistochemistry was done in an Autostainer (Dako). Primary antibodies used were WT1 (clone 6 F-H2, RTU, 1:1, Dako), PTEN (clone 6H2.1, 1:100, Dako), p53 (clone DO-7, RTU, 1:1, Dako) and Ki-67 (clone MIB-1, RTU, 1:1, Dako). Amplification and visualisation of immune complexes and counterstaining were also performed in the Autostainer with the use of an EnVision FLEX+, Mouse (Dako, CA, USA). Slides were counterstained with hematoxylin and evaluated by two independent pathologists.
3
Immunohistochemical Analysis of EZH2 and p53
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Paraffin blocks of CAM with grafted tumors were cut into 3 µm slices and then processed using standard deparaffinization and rehydration techniques. The polyclonal anti-KMT6/EZH2 (phospho S21, ab84989, Abcam) and monoclonal anti-p53 (aa 211-220, clone240, CBL404, Millipore) antibodies were used as the primary antibodies to detect positively stained U87 cells. The primary antibody was detected using biotinylated secondary antibody (DAKO EnVision Flex + Mouse) followed by horseradish peroxidase-conjugated streptavidin (DAKO EnVision Flex/HRP) used as recommended by the manufacturer. Finally, positive reactions were visualized using a 3,3′-diaminobenzidine chromogen (DAB, DAKO, Glostrup, Denmark). After incubation in chromogen, the slides were counterstained with haematoxylin, dehydrated, cleared in xylene, and mounted with a mounting medium. In every formed tumor, EZH2 and p53 positive cells were counted. In every tumor, two equal fields were randomly selected (size of the field 10000 µm2). In every field, all cells were counted, positively stained cells and calculated percentage of EZH2 and p53 cells. Data were presented as % of positively stained cells in every group.
4
Immunohistochemical Analysis of Lung Tissue
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Lung samples were fixed in formaldehyde, embedded in paraffin and serial sections were prepared for cell (alpha-smooth muscle actin-α-sma cells) and ECM glycoprotein staining. Tissue sections were deparaffinized, rehydrated and then antigen was retrieved by being boiled in Tris-EDTA buffer (pH 9.0) for 5 min. Samples were blocked with 0,3% H2O2 for 1 h with avidin/biotin blocking kit (vector). Immunohistochemistry was carried out using primary antibody of tenascin C (1/300, Abcam), versican (1/250, Lifespan) and fibronectin domain EDA (1/400, Abcam), followed by biotin-avidin/peroxidase (Vectastain Elite ABC kit) incubation. Alpha-smooth muscle actin (α-sma) (RTU Flex, Dako) was performed with Autostainer Link Instrument, using EnVision Flex+, mouse, and high pH (Link) (Dako), according to the manufacturer’s recommendation. All sections were counterstained with haematoxylin.
5
Immunohistochemical Characterization of Breast Tumors
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The intrinsic subtype was immunohistologically identified via ready-to-use antibodies for the estrogen-receptor protein (monoclonal rabbit anti-human estrogen receptor α, clone EP1, code IR084, Dako) progesterone-receptor protein (monoclonal mouse anti-human progesterone receptor, clone PgR 636, code IR068, Dako), Her2 (polyclonal rabbit anti-human c-erbB-2 oncoprotein, code A0485, Dako) and Ki-67 (monoclonal mouse anti-human Ki-67 antigen, clone MIB-1, code IR626, Dako). For the horse-radish based peroxidase detection, EnVision Flex Peroxidase-Blocking Reagent (DAKO, SM801), EnVision Flex+ Rabbit (LINKER) (DAKO, K8019) or EnVision Flex+ Mouse (LINKER) (DAKO, K8021) and EnVision Flex/HRP (DAKO, SM802) were used. Counterstaining was performed with hematoxylin before adding the coverslip.
As an internal positive control, patient-derived, non-neoplastic mammary glands were used for ER, PR and Ki-67 (nuclear staining). For Her2, tissue specimens from Her2 positive breast cancer patients (score 3 according to Wolff et al.68 (link)) were included for every Her2 staining session as external positive control.
As an internal positive control, patient-derived, non-neoplastic mammary glands were used for ER, PR and Ki-67 (nuclear staining). For Her2, tissue specimens from Her2 positive breast cancer patients (score 3 according to Wolff et al.68 (link)) were included for every Her2 staining session as external positive control.
6
Immunohistochemical Staining of TMAs
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From the TMAs 2 µm-thick sections were cut and mounted onto glass slides. All glass slides were stored for 2 days at 58°C at the drying chamber, deparaffinized using xylene and dehydrated with ethanol. Subsequently all TMAs were stained using ready to use antibodies for vimentin (monoclonal mouse anti-Vimentin, DAKO, clone V9, code IR630), cytokeratin 7 (monoclonal mouse anti-human Cytokeratin 7, DAKO, clone OV-TL, code IR61961) and histone H2A (monoclonal rabbit anti-human H2A.X(D17A3)XP, CellSignaling, code 7631). Host dependent Streptavidin–biotin based peroxidase detection was performed using the EnVision® Flex Peroxidase-Blocking Reagent (DAKO, SM801), EnVision® Flex + Rabbit (LINKER) (DAKO, SM804) or EnVision® Flex + Mouse (LINKER) (DAKO, SM805) and EnVision® Flex/HRP (DAKO, SM802). Counterstaining was performed with hematoxylin before adding a cover slip. For external positive controls, tissue specimens derived from the colon, placenta and kidney were added onto the TMA.
7
Histological Analysis of Cell Aggregates
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The CAMA was covered with 2% formalin solution in PBS overnight. To avoid loss of cell aggregates during further processing, microwells were sealed with warm 2.4% (w/v) low melting point agarose. Following paraffin embedding and casting into a block, 2 μm thick sections were cut and mounted onto slides. All slides were stored for two days at 58 °C in a drying chamber, deparaffinized (using xylene) and hydrated (using ethanol). Subsequent slides were stained with haematoxylin and eosin (H&E) according to routine protocols or subjected to immunohistochemistry (IHC). IHC was performed using ready-to-use antibodies for MIB-1 (monoclonal mouse anti-human antigen, Ki-67, Code IR626, MIB-1, Dako), pancytokeratin (monoclonal mouse anti-human antigen, Cytokeratin, Code M0821, MNF116, Dako), E-cadherin (monoclonal mouse anti-human antigen, E-cadherin, Code IR059, NCH-38, Dako), and vimentin (monoclonal mouse anti-human antigen, vimentin, Code IR630, V9, Dako).
For the detection of horseradish peroxidase EnVision Flex peroxidase-blocking reagent (DAKO, SM801), EnVision Flex + mouse (LINKER) (DAKO, K8021) and EnVision Flex/HRP (DAKO, SM802) were used. Before adding the coverslip, haematoxylin counterstaining was performed.
For the detection of horseradish peroxidase EnVision Flex peroxidase-blocking reagent (DAKO, SM801), EnVision Flex + mouse (LINKER) (DAKO, K8021) and EnVision Flex/HRP (DAKO, SM802) were used. Before adding the coverslip, haematoxylin counterstaining was performed.
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