Tumor samples were fixed in 4% paraformaldehyde at 4°C overnight and embedded in paraffin. For HE staining, sections were deparaffinized and then stained with hematoxylin for 8 minutes. After washing with flowing water, they were stained with eosin for 2 minutes. For immunohistochemistry, sections were pre-treated with heat mediated antigen retrieval with Dako Target Retrieval Solution (Ph6.0) (DakoCytomation, Copenhagen, Denmark). After the peroxidase-blocking with Dako peroxidase-blocking solution (DAKO), the sections were incubated with anti-mouse CD4 monoclonal antibody (Sino Biological, Beijing, China) or anti-mouse CD8 polyclonal antibody (Bioss Antibodies, Boston, MA, USA) or Anti-ITGAX rabbit polyclonal antibody (OriGene Technologies, Rockville, MD, USA) overnight. Afterwards, Dako Envision TM+ system, HRP (DAKO) was used for secondary antibody and visualization.
Dako peroxidase blocking solution
Manufactured by Agilent Technologies
Sourced in China, Germany, United States, Canada
Dako peroxidase-blocking solution is a laboratory product used to block endogenous peroxidase activity in tissue samples. It is a key component in immunohistochemistry protocols to prevent non-specific staining.
Automatically generated - may contain errors
Lab products found in correlation
Liquid dab substrate chromogen system, by Agilent Technologies (1 mentions)
Ventana benchmark ultra, by Roche (1 mentions)
Dako antibody diluent, by Agilent Technologies (1 mentions)
X0902, by Agilent Technologies (1 mentions)
K3468, by Agilent Technologies (1 mentions)
Af835, by R&D Systems (1 mentions)
Envision kit, by Agilent Technologies (1 mentions)
Avidin biotin blocking kit, by Vector Laboratories (1 mentions)
Vectamount mounting medium, by Vector Laboratories (1 mentions)
Hematoxylin solution, by Merck Group (1 mentions)
Biotinylated goat anti rabbit igg, by Vector Laboratories (1 mentions)
Nanozoomer 2.0 rs system, by Hamamatsu Photonics (1 mentions)
5 protocols using dako peroxidase blocking solution
1
Immunohistochemical Analysis of Tumor Infiltrating Immune Cells
2
Immunohistochemical Analysis of FFPE Tissues
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For immunohistochemical analyses, two µm thick histological sections were cut from formalin-fixed, paraffin-embedded (FFPE) tissue blocks. Sections were incubated for 30 min at a 70 °C temperature. Afterward, slides were deparaffinized through a series of xylene and gradient alcohols to water. For antigen retrieval, slides were cooked for 8 min at 110 °C in citrate pH 6.0 and then placed in iced water. Next, slides were incubated in 1× Dako Peroxidase-Blocking Solution® (S2023; Dako GmbH, Jena, Germany) for 10 min. The primary antibody was diluted in Dako Antibody Diluent® (S2022). Sections were incubated herein in a humidity chamber at room temperature overnight (see Table 2 for a list of employed antibodies). After washing with Dako washing solution® (S3006), the secondary antibody Histofine Simple Stain MAX PO® anti-rabbit or anti-goat was administered for 60 min at room temperature. Following two additional washes in Dako washing solution® (S3006), the chromogenic reaction was performed with the Dako Liquid DAB + Substrate Chromogen System®. Slides were stained in Mayer’s hemalum for 10 s. Coverslips were automatically placed on top with the Ventana BenchMark Ultra® (Roche, Basel, Switzerland). β-catenin antibodies used with respective dilutions are listed in Table 1 .
3
Immunohistochemical Analysis of Intestinal Samples
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Two commercial antibodies were used as previously described [46 (link),47 (link)]. Briefly, four-micrometer paraffin-embedded transverse sections from formalin-fixed jejunum specimens were dewaxed in toluene and rehydrated by an acetone bath then deionized water. Antigen retrieval was performed in 10 mM citrate buffer pH 6.0 for 30 min in a water bath at 95 °C. Cooled sections were then incubated in Dako peroxidase blocking solution (Dako S2023) to quench endogenous peroxidase activity. Non-specific binding was blocked by incubation in normal goat serum (dilution 1:10, Dako X0902) for 20 min at room temperature. The primary antibodies were anti-Ki-67antigen (Dako M7240, dilution 1:50) and anti-active caspase-3 (R&D system, AF835, dilution 1:300). Sections were incubated with primary antibodies for 50 min at room temperature (RT). Bound primary antibodies were detected with EnVision™ + Horse Radish Peroxydase (HRP) Systems (Dako, K4061) 30 min at RT. Peroxidase activity was revealed by 3,3′-diaminobenzidine tetrahydrochloride substrate (Dako K3468). Finally, sections were counterstained with Harris hematoxylin, dehydrated and coverslipped.
4
Immunohistochemical Evaluation of DOG1 in Pancreatic Cancer
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TMA sections were freshly cut, processed and stained the same day. Slides were deparaffinized with xylol, rehydrated and exposed to heat-induced antigen retrieval. Endogenous peroxidase activity was blocked with Dako Peroxidase Blocking Solution (#52023; Agilent, Santa Clara, CA, USA) for 10 min. Anti-DOG1 mouse monoclonal antibody MSVA-201M (MS Validated Antibodies, Hamburg, Germany) was applied at 37 °C, 60 min, at 1:150. Staining was visualized with the EnVision Kit (#K5007; Agilent, Santa Clara, CA, USA) and counterstained with Haemalaun. DOG1 staining was predominantly membranous in pancreatic cancer cells. Scoring of the staining intensity was semi-quantitatively assessed as previously described in Juhnke et al. (2017) (link). Specifically, four grades were defined: Negative (no staining at all), weak (staining intensity of 1+ in ≤ 70% of the tumor cells or staining intensity of 2+ in ≤ 30% of the tumor cells), moderate (staining intensity of 1+ in >70% of the tumor cells, staining intensity of 2+ in >30% but in ≤ 70% of the tumor cells or staining intensity of 3+ in ≤ 30% of the tumor cells), strong (staining intensity of 2+ in >70% of the tumor cells or staining intensity of 3+ in >30% of the tumor cells).
5
Immunohistochemical Localization of GLUT2 in Mouse Jejunum
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Male P2rx7+/+ and P2rx7−/− mice were starved for 6 h prior to experiments. Jejunum sections (5 μm) were deparaffinized, rehydrated and treated with Dako peroxidase blocking solution (Agilent Technologies Canada, Inc., Mississauga, ON). Jejunum slides were treated with the avidin/biotin blocking kit (Vector laboratories, Brockville, ON) and non-immunogenic sites blocked with PBS containing 0.1% Tween 20 and 2% BSA (PBT-BSA). Slides were incubated with goat polyclonal anti-GLUT2 (C-19) (Santa Cruz Biotech, Santa-Cruz, CA) antibodies at a dilution of 1:100 in PBS-BSA for 2 h at room temperature (RT). Slides were washed in PBS and incubated with biotinylated rabbit anti-goat IgG (1:200; Vector laboratories) in PBT-BSA for 1 h at RT followed by incubation with the Vectastain biotinylated horseradish Peroxydase H Elite ABC Kit (Vector laboratories) and revealed with the Dako DAB and chromogen solution (Agilent Technologies Canada, Inc.). Slides were counterstained with a hematoxylin solution following the manufacturer instructions (Sigma-Aldrich, Oakville, ON) and mounted using VectaMount mounting medium (Vector laboratories). Slides were scanned at 40 X with Hamamatsu Nanozoomer 2.0-RS system and analysed with the NDP scan software.
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