Tumor xenograft cryo-sections were fixed in 4% PFA, treated with 0.4% pepsin in 0.2 N HCl, 3% H2O2–PBS, and then permeabilized and assayed for WT-1 (Dako) expression with anti-mouse EnVision-HRP (Dako). Calretinin antibody (Dako) was used on sections fixed in 4% PFA, followed by heat-induced antigen retrieval and treatment with 0.3% H2O2–PBS and permeabilization. Detection was performed using streptavidin/horseradish peroxidase (Dako). Mesothelin staining was assessed using a secondary antibody conjugated with a green fluorescent dye. For D2-40 staining (Dako), heat antigen retrieval was performed using Target Retrieval Solution S1700 (Dako) and permeabilization in PBS 10% NGS 0.3% Triton X-100. Mouse IgG and the omission of primary antibody were used as negative controls [25 (link)]. Digital images were captured by a Nikon Eclipse microscope (Nikon Europe).
Anti mouse envision hrp
Manufactured by Agilent Technologies
Sourced in United States
The Anti-mouse EnVision-HRP is a laboratory equipment used for immunohistochemistry applications. It is a polymer-based detection system that utilizes horseradish peroxidase (HRP) enzyme for signal amplification. The product is designed to bind to mouse primary antibodies and allows for the visualization of target antigens in tissue samples.
Automatically generated - may contain errors
Lab products found in correlation
Eclipse microscope, by Nikon (1 mentions)
Streptavidin horseradish peroxidase, by Agilent Technologies (1 mentions)
Target retrieval solution s1700, by Agilent Technologies (1 mentions)
Ab52472, by Abcam (1 mentions)
Ki 67 clone mib 1, by Agilent Technologies (1 mentions)
Anti cleaved caspase 3 asp175, by Cell Signaling Technology (1 mentions)
Sc 53993, by Santa Cruz Biotechnology (1 mentions)
Anti γh2ax, by Cell Signaling Technology (1 mentions)
Opal 520, by PerkinElmer (1 mentions)
Nat105, by Biocare Medical (1 mentions)
Envision anti rabbit hrp, by Agilent Technologies (1 mentions)
Tsa cy5, by PerkinElmer (1 mentions)
E1l3n, by Cell Signaling Technology (1 mentions)
Tsa cy3, by PerkinElmer (1 mentions)
Lsm780 confocal microscope, by Leica camera (1 mentions)
Dmi6000b microscope, by Leica camera (1 mentions)
3 3 diaminobenzidine (dab), by Agilent Technologies (1 mentions)
Ultra fast scanner 1.6 ra, by Philips (1 mentions)
Haematoxylin, by Thermo Fisher Scientific (1 mentions)
Citrate, by Agilent Technologies (1 mentions)
9 protocols using anti mouse envision hrp
1
Immunohistochemical Analysis of Tumor Xenografts
2
Quantitative Immunohistochemical Analysis of Autophagy Markers
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The slides were incubated with the primary antibodies (1:100 dilution, rabbit monoclonal antibody (mAb) for ATG7, ab 52472, Abcam, UK; 1:500 dilution, rabbit mAb for LC3, 3868 CST, USA; 1:200 dilution, rabbit mAb for Beclin-1, 3395 CST, USA; and 1:75 dilution, ki-67, clone MIB-1, Dako, Santa Clara, CA, US) overnight at 4 °C and with Anti-rabbit Envision+/horseradish peroxidase (HRP) (Dako) or Anti-mouse Envision+/HRP (Dako) secondary antibody for 1 h. Staining of ATG-7, LC3, and Beclin-1 was categorised semi-quantitatively based on the percentage of positive tumour cells: 0 (5% positive cells), 1 (6–25% positive cells), 2 (26–50% positive cells), 3 (51–75% positive cells), and 4 (>75% positive cells). Cytoplasmic and membrane staining intensities were also determined semi-quantitatively as follows: 0 (negative), 1 (weakly positive), 2 (moderately positive), and 3 (strongly positive). Sections’ scores were defined as the extent of staining × intensity, while the Ki-67 score was calculated as the percentage of positively stained cells among the total number of malignant cells scored. The scoring was conducted using three high-power (×400) fields in the invasive edge of the tumour, which represents the spectrum of staining observed in the whole section.
3
Immunohistochemical Analysis of Cellular Markers
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Formalin-fixed and paraffin-embedded tissue sections were deparaffinized in xylene and graded alcohols, hydrated, and washed in a phosphate-buffered saline (PBS). After antigen retrieval in a sodium citrate buffer (pH 6) in a microwave oven, the endogenous peroxidase was blocked by 0.3% H2O2 for 15 min. Sections were incubated overnight at 4 °C with primary antibodies anti-γ-H2A.X (#9718 Cell Signal Technology, MA, USA), anti-cleaved Caspase-3 (Asp175) (Cell signaling Technology, MA, USA) or anti-Ki-67 (SP6, Neomarkers, CA, USA), respectively. As a secondary antibody, the anti-rabbit-horseradish peroxidase (HRP) SignalStain Boost IHC detection kit was used (# 8114, Cell Signaling Technology, MA, USA). For the assessment of MycN expression, a monoclonal mouse anti-MycN antibody was used (sc-53993, Santa Cruz Biotechnology, CA, USA). As a secondary antibody, anti-mouse EnVision-HRP (Dako, Agilent Technologies, Inc., Santa Clara, CA, USA) was used. A matched isotype control was also used as a control for nonspecific background staining.
4
Multiplexed Immunohistochemistry for PNS Biomarkers
5
Immunohistochemical Evaluation of MCPyV, IL-33, and Related Markers
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Formalin-fixed and paraffin-embedded tissue sections were deparaffinized in xylene and graded alcohols, and then hydrated and washed in PBS. After antigen retrieval in a sodium citrate buffer (pH 6) in a microwave oven, the endogenous peroxidase was blocked by 0.3% H2O2 for 15 min. Sections were incubated overnight at 4 °C with the primary antibodies against MCPyV−LT, IL-33, ST2/IL1RL1, IL1RAcP, and CK20 (Supplementary Table S1 ). As a secondary antibody, either the anti-rabbit-HRP Signalstain (R) DAB Substrate kit (Cat.# 8059S Cell Signaling Technologies, Beverly, MA, USA) or anti-mouse EnVision-HRP (Dako, Agilent Technologies, Inc., Santa Clara, CA, USA) was used. A matched isotype control was used as a control for nonspecific background staining. Staining intensity was scored as negative, low, intermediate, or strong when >20% of tumor cells showed protein expression.
6
Immunohistochemical Analysis of CCR4, CCL17, MCPyV-LT, and CK20 in Tissue Sections
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Formalin-fixed and paraffin-embedded tissue sections were deparaffinized in xylene and graded alcohols, hydrated and washed in PBS. After antigen retrieval in a sodium citrate buffer (pH 6) in a microwave oven, the endogenous peroxidase was blocked by 0.3% H2O2 for 15 min. Sections were incubated overnight at 4°C with the primary antibody CCR4 (Abcam Cat.#ab1699, Cambridge, UK), CCL17/TARC (Abcam Cat.#ab182793), MCPyV-LT (Santa Cruz Biotechnology Cat.#sc-136172) and CK20 (Roche Cat.#790-4431). As a secondary antibody, the anti-rabbit-HRP SuperPicTure Polymer detection kit (87-9663, Zymed-Invitrogen, San Francisco, CA, USA) or anti-mouse EnVision-HRP (Dako, Agilent Technologies, Inc., Santa Clara, CA, USA) was used. A matched isotype control was used as a control for nonspecific background staining.
7
Immunohistochemistry and Immunofluorescence Tissue Staining
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Formalin-fixed and paraffin-embedded tissue sections were deparaffinized in xylene and graded alcohols, hydrated and washed in PBS. After antigen retrieval in a sodium citrate buffer (pH 6) in a microwave oven, the endogenous peroxidase was blocked by 0.3% H2O2 for 15min. Sections were incubated overnight at 4°C with the primary antibody. As a secondary antibody, the anti-rabbit-HRP SuperPicTure Polymer detection kit (87-9663, Zymed-Invitrogen, San Francisco, USA) or anti-mouse EnVision-HRP (Dako, Agilent Technologies, Inc., Santa Clara, USA) was used. A matched isotype control was used as a control for nonspecific background staining.
For immunofluorescence histology studies (IF-P), the sections were treated as described above and stained sequentially with the primary and secondary antibody sets. Alexa Fluor® 488 and Alexa Fluor® 594 conjugated secondary antibodies were used to visualize positive staining.
The fluorescence labeled tissue sections were examined using the Zeiss LSM780 confocal microscope and the immunoperoxidase stained sections using the Leica DMI6000B microscope.
For immunofluorescence histology studies (IF-P), the sections were treated as described above and stained sequentially with the primary and secondary antibody sets. Alexa Fluor® 488 and Alexa Fluor® 594 conjugated secondary antibodies were used to visualize positive staining.
The fluorescence labeled tissue sections were examined using the Zeiss LSM780 confocal microscope and the immunoperoxidase stained sections using the Leica DMI6000B microscope.
8
Immunohistochemical Staining of CEA in FFPE Tissue
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Immunohistochemical staining for CEA was performed on FFPE tissue. Tissue samples were stained using a monoclonal mouse antibody against CEACAM5 (clone number CI-P83-1; Santa Cruz Biotechnology). Validation of the staining protocol was performed using human colon cancer tissue as a positive control. Sections (thickness: 4 µm) were cut from paraffin blocks and mounted on adhesive slides (Starfrost). Slides were deparaffinized using xylene and rehydrated in decreasing concentrations of ethanol. Subsequently, slides were rinsed with distilled water, and endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide (Merck Millipore) for 20 minutes. Antigen retrieval was performed in DAKO PT Link with target retrieval solution pH 6.0 at 95°C for 10 minutes. After rinsing with phosphate-buffered saline (PBS) the primary antibody was incubated overnight at room temperature and afterwards rinsed with PBS. Incubation of the secondary antibody (Envision anti-mouse HRP; Dako) was performed for 30 minutes at room temperature, followed by rinsing with PBS. Antibody binding was then visualized using 3,3′-diaminobenzidine (DAKO). Sections were counterstained with hematoxylin (Klinipath), rinsed in tap water, dehydrated, and mounted with Pertex. All slides were scanned using the Philips Ultra Fast Scanner 1.6 RA.
9
Immunohistochemical Analysis of Psoriasin in Psoriatic Skin
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Skin punch biopsies were obtained from healthy controls and lesional psoriasis skin and were fixed and paraffin-embedded. The sections were deparaffinized in Histolab-clear (Histolab Products, Gothenburg, Sweden) and rehydrated in ethanol. Heatinduced antigen retrieval was performed in citrate (pH = 6) antigen retrieval buffer (DAKO, Glostrup, Denmark). Endogenous peroxidases were blocked with 3% hydrogen peroxide, and unspecific protein binding was inhibited with 5% bovine serum albumin. Antibody binding with primary mouse antihuman psoriasin antibody (1:400; Imgenex, San Diego, CA, USA) was carried out overnight at 4°C. Following incubation, DAKO EnVision antimouse HRP conjugated polymer was applied, and antigenantibody complexes were visualized by DABstaining (PolySciences, Eppelheim, Germany). Cell nuclei were counterstained with haematoxylin (Invitrogen, Paisley, UK).
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