Immunohistochemistry was performed using the IHC Staining Kit (Dako, Santa Clara, CA, USA). Tissue sections were deparaffinized in xylene (Thermo), hydrated in phosphate‐buffered saline (Welgene), and blocked with Background Reducing Solution (Dako). The sections were incubated with anti‐CASQ2 (#NBP1‐87304; NOVUS), anti‐aSMA (#BS70000; Bioworld Technology), anti‐FSP1 (#BS7671; Bioworld Technology), anti‐HIF1α (#NB100‐131; NOVUS), anti‐vimentin (#5741; Cell Signaling Technology), anti‐pan‐cytokeratin (#M3515; Dako), and anti‐ki67 (#9027; Cell Signal Technology) at 4 °C overnight, followed by incubation with horseradish peroxidase‐conjugated anti‐secondary antibody (Dako). The signal was developed using diaminobenzidine and hydrogen peroxide, resulting in a brown precipitate. The sections were counterstained with hematoxylin (Dako), dehydrated, and mounted. The DAB area was quantified using IHC Toolbox in ImageJ software (NIH) with 20 random histological fields from five slides of tumor tissues per group [22 (link)].
Background reducing solution
Manufactured by Agilent Technologies
Sourced in United States
The Background Reducing Solution is a laboratory reagent designed to reduce background signals in analytical techniques. It is a clear, colorless liquid that can be added to samples or used in the preparation of reagents to help improve the signal-to-noise ratio and enhance the detection of target analytes.
Automatically generated - may contain errors
Lab products found in correlation
Xylene, by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by Welgene (1 mentions)
Hematoxylin, by Agilent Technologies (1 mentions)
Ihc staining kit, by Agilent Technologies (1 mentions)
Anti pan cytokeratin, by Agilent Technologies (1 mentions)
Anti ki67, by Cell Signaling Technology (1 mentions)
Anti vimentin, by Cell Signaling Technology (1 mentions)
Anti hif 1α, by Novus Biologicals (1 mentions)
Target retrieval solution, by Agilent Technologies (1 mentions)
Clone jc70a, by Agilent Technologies (1 mentions)
Csaii kit, by Agilent Technologies (1 mentions)
Mayer s hematoxylin, by Avantor (1 mentions)
3 protocols using background reducing solution
1
Comprehensive Immunohistochemistry Protocol
2
Immunohistochemical Analysis of CD31 Expression
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Immunohistochemical determination of CD31 expression (platelet endothelial cell adhesion molecule-1, PECAM-1) was conducted as this is a marker protein of mature vascular endothelium [19 (link)]. Sections (5 μm) were mounted on chromalum-coated slides, dewaxed, rehydrated, rinsed, and washed in PBS 1x solution for 30 min. Once endogenous peroxidase was quenched, the specimens were treated with target retrieval solution (Dako) equilibrated at 99°C. Tissue samples were then incubated for 40 min with 1/50 dilution of anti-CD31 antibody (Abcam 28364, USA) in background reducing solution (Dako). The immunohistochemical reactions were carried out using the labelled streptavidin/biotin-horseradish peroxidase conjugate method, according to the manufacturer's instructions (Dako). The peroxidase reaction was developed with diaminobenzidine and counterstained with hematoxylin.
3
Immunohistochemical Evaluation of Vascular Markers
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Sections from all the patients in the CON group and sections from 10 random samples from the BML group were deplastified in 2-methoxyethyl acetate and washed in ethanol. Epitopes were retrieved using antigen retrieval solutions based on the manufacturer's (Dako, CA, USA) recommendations for each primary antibody. For the rest of the immunostaining procedure CSA II Kit (Dako) was used according to the manufacturer's instructions.
We used monoclonal mouse anti-human IgG antibodies against von Willebrand Factor (vWF, Clone F8/86, Dako) and CD31 (Clone JC70A, Dako) as primary antibodies in the setup. Sections were incubated overnight at 4 C with the primary antibodies diluted in a background reducing solution (1:25) (Dako), washed three times in TBS between each step before signal detection with diaminobenzidine (Dako) and counter-staining with Mayer's hematoxylin (VWR, PA, USA). Mouse IgG1 antibody against Aspergillus niger glucose oxidase (X0931, Dako) was used on both BML and CON sections as negative control. For positive controls, sections of human lung and tonsil tissue were used.
We used monoclonal mouse anti-human IgG antibodies against von Willebrand Factor (vWF, Clone F8/86, Dako) and CD31 (Clone JC70A, Dako) as primary antibodies in the setup. Sections were incubated overnight at 4 C with the primary antibodies diluted in a background reducing solution (1:25) (Dako), washed three times in TBS between each step before signal detection with diaminobenzidine (Dako) and counter-staining with Mayer's hematoxylin (VWR, PA, USA). Mouse IgG1 antibody against Aspergillus niger glucose oxidase (X0931, Dako) was used on both BML and CON sections as negative control. For positive controls, sections of human lung and tonsil tissue were used.
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