Tissue sections of eight-micrometer thickness were taken from paraffin-embedded blocks using a standard microtome (Leica SM2000, Wetzlar, Germany) and mounted onto microscope slides for analysis. For heat-induced antigen retrieval, after dewaxing and rehydration, all slides were subjected to microwave treatment (3×5 min each, 600 W, in 10 mM citrate buffer, pH 6.0). The slides were then immersed into 3% H2O2 and 100% methanol in order to block endogenous peroxidase activity. Then, the slides were subjected to overnight incubation at 4 °C with mouse monoclonal anti-fascin antibody (1:50; Dako) and treated with biotinylated anti-mouse secondary antibody (1:100; DAKO). Finally, the slides were incubated with streptavidin-peroxidase (1:100; Dianova), and DAB/H2O2 (1.85 mM). PBS was used for all washing procedures. Haematoxylin was used for counterstaining the slides. As negative control, PBS was used instead of primary antibody. In addition, the endothelium tissues were considered as positive control of staining.
Streptavidin peroxidase
Manufactured by Dianova
Sourced in Germany
Streptavidin-peroxidase is a conjugated protein composed of streptavidin and horseradish peroxidase. Streptavidin has a high affinity for biotin, while peroxidase is an enzyme that catalyzes oxidation-reduction reactions.
Automatically generated - may contain errors
2 protocols using streptavidin peroxidase
1
Immunohistochemical Analysis of Fascin Expression
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Tissue sections of eight-micrometer thickness were taken from paraffin-embedded blocks using a standard microtome (Leica SM2000, Wetzlar, Germany) and mounted onto microscope slides for analysis. For heat-induced antigen retrieval, after dewaxing and rehydration, all slides were subjected to microwave treatment (3×5 min each, 600 W, in 10 mM citrate buffer, pH 6.0). The slides were then immersed into 3% H2O2 and 100% methanol in order to block endogenous peroxidase activity. Then, the slides were subjected to overnight incubation at 4 °C with mouse monoclonal anti-fascin antibody (1:50; Dako) and treated with biotinylated anti-mouse secondary antibody (1:100; DAKO). Finally, the slides were incubated with streptavidin-peroxidase (1:100; Dianova), and DAB/H2O2 (1.85 mM). PBS was used for all washing procedures. Haematoxylin was used for counterstaining the slides. As negative control, PBS was used instead of primary antibody. In addition, the endothelium tissues were considered as positive control of staining.
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Tissue sections of eight-micrometer thickness were taken from paraffin-embedded blocks using a standard microtome (Leica SM2000, Wetzlar, Germany) and mounted onto microscope slides for analysis. For heat-induced antigen retrieval, after dewaxing and rehydration, all slides were subjected to microwave treatment (3×5 min each, 600 W, in 10 mM citrate buffer, pH 6.0). The slides were then immersed into 3% H2O2 and 100% methanol in order to block endogenous peroxidase activity. Then, the slides were subjected to overnight incubation at 4 °C with mouse monoclonal anti-fascin antibody (1:50; Dako) and treated with biotinylated anti-mouse secondary antibody (1:100; DAKO). Finally, the slides were incubated with streptavidin-peroxidase (1:100; Dianova), and DAB/H2O2 (1.85 mM). PBS was used for all washing procedures. Haematoxylin was used for counterstaining the slides. As negative control, PBS was used instead of primary antibody. In addition, the endothelium tissues were considered as positive control of staining.
2
Immunoblotting Protocol for Detecting Ductin in Ovarian Tissue
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Immunoblots were performed as described previously [11 (link)]. In short, homogenates of ovaries were sonicated and briefly boiled. The proteins were separated using 12 % SDS-PAGE and transferred to nitrocellulose membranes. Nonspecific binding sites were blocked with 5 % skimmed milk powder/PBS and the blots were incubated in Anti-ductin diluted 1:100 up to 1:500 with 1 % BSA/PBS. NIS diluted 1:200 was used as a control [11 (link)]. Subsequently, biotinylated goat-anti-rabbit IgG (Jackson, PA, USA; diluted 1:1000), streptavidin-peroxidase (Dianova, Germany; diluted 1:1000) and H2O2/4-chloro-1-naphthol (Sigma, Germany) were applied, and photographs were taken using a digital camera. Experiments were performed three times.
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