Tissue blocks were sectioned at 4 μm on a graded slide, deparaffinized in xylene, rehydrated in graded ethanols, and rinsed in phosphate-buffered saline (PBS). Immunohistochemistry (IHC) was performed on a DakoCytomation autostainer using the Envision HRP Detection system (Dako, Carpinteria, CA). Each mammary tissue block was sectioned at 4 μm on a graded slide, deparaffinized in xylene, rehydrated in graded ethanols, and rinsed in Tris-phosphate-buffered saline (TBS). Heat induced antigen retrieval was performed in a microwave at 98 °C in 0.01 M citrate buffer. After cooling for 20 min, sections were rinsed in TBS and subjected to the primary mouse monoclonal anti-CD163 [GH1/61] antibody (1:100, Abcam, ab111250) or the primary rabbit polyclonal anti-HLA-DR antibody (1:250, Abcam, ab137832) for 30 min. Immunoreactivity was visualized by incubation with chromogen diaminobenzidine (DAB) for 5 min. Tissue sections were counterstained with hematoxylin, dehydrated through graded ethanols and xylene, and cover-slipped. Images were captured with an Olympus BX41 light microscope using SPOT Software 5.1 (SPOT™ Imaging Solutions, Detroit, MI).
Envision hrp detection system
Manufactured by Agilent Technologies
Sourced in United States
The EnVision-HRP detection system is a laboratory equipment designed for the detection and quantification of target analytes in various assays. It utilizes a horseradish peroxidase (HRP) based signal amplification system to enhance the sensitivity of the detection process. The core function of the EnVision-HRP detection system is to provide a reliable and sensitive method for the analysis of samples in a laboratory setting.
Automatically generated - may contain errors
Lab products found in correlation
Asp300s processor, by Leica (2 mentions)
Trs high ph, by Agilent Technologies (2 mentions)
Ab111250, by Abcam (1 mentions)
Automated immunostainer, by Agilent Technologies (1 mentions)
Streptavidin horseradish peroxidase, by BD (1 mentions)
Bcl 2 oncoprotein, by Agilent Technologies (1 mentions)
Peroxidase blocking solution, by Agilent Technologies (1 mentions)
Liquid dab chromogen, by Agilent Technologies (1 mentions)
Target retrieval solution, by Agilent Technologies (1 mentions)
Poly l lysine coated glass slides, by Matsunami (1 mentions)
Background reducing diluent, by Agilent Technologies (1 mentions)
Dako antibody diluent, by Agilent Technologies (1 mentions)
Dab chromogen, by Agilent Technologies (1 mentions)
Envision detection system, by Agilent Technologies (1 mentions)
8 ohdg, by Abcam (1 mentions)
Nf kb p65, by Abcam (1 mentions)
Fisherbrand superfrost plus slides, by Thermo Fisher Scientific (1 mentions)
Lc3a b, by Abcam (1 mentions)
Hmgb1, by Abcam (1 mentions)
Vcam 1, by Abcam (1 mentions)
16 protocols using envision hrp detection system
1
Immunohistochemistry Protocol for CD163 and HLA-DR
2
Immunostaining of BM biopsies for NK cells
3
Immunohistochemistry Protocol for Tissue Sections
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Tissues were fixed in 10% buffered formalin for 24 h at 4 °C, after which the tissues were moved to 70% ethanol for at least 24 h before processing. Tissues were processed in a Leica ASP300S processor before being embedded in paraffin. Tissue blocks were sectioned at a thickness of 4 μm and mounted on charged slides. Sections were deparaffinized in xylenes, rehydrated in ethanol, and rinsed in phosphate buffered saline (PBS). Immunohistochemistry (IHC) was performed on a DakoCytomation autostainer, using the envision HRP Detection system (Dako, Carpinteria, CA). Antigen retrieval was performed in a microwave at 98 °C in 0.01 M citrate buffer. After cooling for 20 min, sections were rinsed in TBS and incubated with primary monoclonal antibodies (Table 1 ) for 30 min. After a subsequent wash in TBS, slides were incubated with corresponding secondary antibody (Table 1 ) for 20 min. Immunoreactivity was visualized by incubation with the chromogen diaminobenzidine (DAB) for 5 min. Tissue sections were counterstained with hematoxylin, dehydrated through graded ethanol and xylenes, and then cover slips were applied. Images were captured with an Olympus BX41 light microscope using SPOT Software 5.1 (SPOT™ Imaging Solutions, Detroit, MI) [48 (link)].
4
Immunohistochemical Staining of CD8+ T Cells
5
Immunohistochemical Staining for Bmi-1, CK15, and Bcl-2
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Immunohistochemical staining was carried out according to a standard method. 3-μm tissue sections were deparaffinised in xylene and rehydrated through a graded alcohol series. Heating in a microwave oven in a target retrieval solution pH 9.0 (TRS High pH; Dako) for 30 min was used for antigen retrieval. Endogenous peroxidase activity was quenched with 0.3% hydrogen peroxide in methanol for 30 min. The sections were washed with TBS and incubated with primary antibodies against: Bmi-1 (Invitrogen, USA, dilution 1 : 600, Catalog number PA5-23308), and CK15 (Invitrogen, USA, dilution 1 : 400, Catalog number MA1-90926), Bcl-2 Oncoprotein (Dako, RTU- Flex, clone 124, Catalog number IR 614). After washing, an adequate EnVision-HRP detection system (Dako, Carpinteria, CA, USA) was used. 3,3’-diaminobenzidine was used as the chromogen. After counterstaining with Mayer’s haematoxylin, the slides were washed, dehydrated, cleared in xylene and coverslipped. Negative controls for immunohistochemical staining were prepared with primary antibodies replaced by the antibody diluent.
6
Immunohistochemistry Protocol for Tissue Sections
7
Immunohistochemical Analysis of EMT Markers
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The antibody sources used in this study are shown in Table 2 . Paraffin sections were cut (3 μm) and mounted on poly-l -lysine–coated glass slides (Matsunami, Tokyo, Japan). Sections were routinely dewaxed and dehydrated and then subjected to heat-induced epitope retrieval in high pH Target Retrieval Solution (Dako, Carpinteria, Calif). The slides were placed in peroxidase-blocking solution (Dako) to inhibit non–specific-binding activity.
Immunostaining for E-cadherin and EMT-related proteins, including Slug, Twist and Zeb1, was performed by placing the sections in a microwave oven for 30 minutes for antigen retrieval. After primary antibody treatment, sections were examined using the EnVision HRP detection system (Dako). The antigen-antibody complex was visualized with DAB+ liquid chromogen (Dako) and counterstained with hematoxylin before mounting. Representative histologic findings and the immunoreactivity of E-cadherin and EMT markers in APC are shown in Figure1 .
Immunostaining for E-cadherin and EMT-related proteins, including Slug, Twist and Zeb1, was performed by placing the sections in a microwave oven for 30 minutes for antigen retrieval. After primary antibody treatment, sections were examined using the EnVision HRP detection system (Dako). The antigen-antibody complex was visualized with DAB+ liquid chromogen (Dako) and counterstained with hematoxylin before mounting. Representative histologic findings and the immunoreactivity of E-cadherin and EMT markers in APC are shown in Figure
8
Quantifying Progesterone Receptor Expression in Mammary Tissue
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Immunohistochemistry (IHC) was performed on a DakoCytomation autostainer using the Envision HRP Detection system (Dako, Carpinteria, CA). Mammary tissue blocks were sectioned at 4 μm, deparaffinized in xylene, rehydrated in graded ethanols, and rinsed in Tris-phosphate-buffered saline (TBS). Heat-induced antigen retrieval was performed in a microwave at 98 °C in 0.01 M citrate buffer. After cooling for 20 min, sections were rinsed in TBS and incubated with rabbit polyclonal anti-PR 1:500, (Cell Signaling; #8757) for 30 min at room temperature. Immunoreactivity was visualized by incubation with diaminobenzidine for 5 min. Tissue sections were counterstained with hematoxylin, dehydrated through graded ethanols and xylene, and cover-slipped. Images were captured with an Olympus BX41 light microscope using (SPOT™Imaging Solutions, Detroit, MI). PR staining of epithelial cells was quantified using ImageJ.
9
Immunohistochemical Staining of SOX2 and TAZ
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Immunohistochemical staining was carried out according to a standard method. 3-µm tissue sections were deparaffinized in xylene and rehydrated through a graded alcohol series. Heating in a microwave oven in a solution of target retrieval solution pH 9.0 (TRS High pH; Dako), for 30 minutes was used for antigen retrieval. Endogenous peroxidase activity was quenched with 0,3% hydrogen peroxide in methanol for 30 minutes. The sections were washed with TBS and incubated all night with polyclonal rabbit primary antibodies against: SOX2 (ThermoFisher Scientific, USA, dilution 1:300, Catalog number PA1-094), and TAZ (Abcam, UK, dilution 1:400, Catalog number ab84927). The sections for α-SMA staining were incubated 30 minutes with monoclonal mouse primary antibodies against actin (Dako; clone: 1A4, RTU) After washing, an adequate EnVision-HRP detection system (Dako, Carpinteria, CA, USA) was used. 3,3'-diaminobenzidine was used as the chromogen. After counterstaining with Mayer's hematoxylin, the slides were washed, dehydrated, cleared in xylene and coverslipped. The negative controls for immunohistochemical staining were prepared with the primary antibodies replaced by the antibody diluent.
10
Immunohistochemical Analysis of PQCL2 in Gastric Cancer
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Gastric cancer tissue samples were fixed in 10% buffered formalin and embedded in paraffin. The blocks were cut into 3‐μm sections for immunohistochemistry using a rabbit polyclonal anti‐PQCL2 Ab and the rabbit EnVision‐HRP detection system (Dako, Carpinteria, CA, USA). After deparaffinization, antigen retrieval was carried out in 10 mmol/L sodium citrate buffer (pH 6.0) using a pressure cooker at full power for 4 minutes. The sections were then treated with 3% hydrogen peroxide for 10 minutes before overnight incubation in a humid chamber at 4°C with the primary Ab diluted 1:100 in background‐reducing diluent (Dako). A rabbit IgG isotype control without the primary Ab was used as a negative control. The sections were incubated with EnVision reagent for 30 minutes followed by diaminobenzidine for 5 minutes and counterstained with Meyer's hematoxylin. At each step, sections were rinsed several times with TBS with 0.3% Tween‐20.
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