The osteosarcoma tissue was embedded in paraffin and cut into 5-μm-thick sections. The sections were then dried overnight at 37 °C, dewaxed in xylene, and rehydrated with graded alcohol. Next, the tissue sections were quenched in 3% hydrogen peroxide, blocked with 5% BSA for 1 h, and immunostained with primary antibody against CD31 (ab182981, 1:2000, Abcam) at 4 °C overnight. After PBS washing, the sections were incubated with the secondary antibody for 1 h. All sections were incubated with Envision system-HRP substrate using a Dako REAL Ebision kit (DAKO, Glostrup, Denmark) before observation under a microscope (Leica). Evaluation of MVD in tumor tissues was implemented referring to a previous study [14 (link)].
Dako real ebision kit
Manufactured by Agilent Technologies
Sourced in Denmark
The Dako REAL Ebision kit is a laboratory equipment product designed for in situ hybridization (ISH) applications. It provides a standardized and efficient method for the detection of specific nucleic acid sequences within tissue sections or cell preparations.
Automatically generated - may contain errors
5 protocols using dako real ebision kit
1
Immunohistochemical Analysis of Osteosarcoma Angiogenesis
2
Immunohistochemical Analysis of Tumor Proliferation
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The tumor tissues were fixed in 4% PFA at 4°C overnight, washed by PBS, and then embedded in paraffin. The tumor tissues were sliced into 5 μm thick sections and dried overnight at 37°C on glass slides. The dried slices were deparaffinized in xylene first followed by rehydration through a graded concentration of alcohol. 3% hydrogen peroxide was used to quench the sections and 5% BSA was added to block the slices for 1 h at room temperature. Primary antibody (anti-Ki-67; 1:1,000; Invitrogen, USA) was added to incubate with the slices at 4°C overnight. The next day, the antibody was washed off by PBS and secondary antibodies were added to incubate with sections for 1 h at room temperature. Substrates of the Envision system-horseradish peroxidase (HRP) from the kit (Dako REAL Ebision kit; DAKO, Glostrup, Denmark) were added to incubate with stained slices as the manufacturer’s protocol described and the signals were analyzed with a light microscope.
3
Immunohistochemical Staining of Tumor Tissues
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Tumor tissue samples were fixed with formalin (10%) and embedded with paraffin. For IHC staining, the samples were treated with 5% BSA at room temperature for 1 h before they were cut into 5 μm sections. Blocked sections were then treated with CD31 (#77699, Cell Signaling Technology, USA) or BMP4 (#12492-1-AP, Proteintech, Beijing, China) primary antibody at 4 °C on a shaker overnight. The sections were washed with PBS twice and incubated at room temperature for 2 h with secondary antibodies. After washing with PBS again, sections were incubated with HRP (Dako REAL Ebision kit; DAKO, Glostrup, Denmark) based on the manufacturer’s protocol.
4
Immunohistochemical Analysis of Cdc25A in Tumor Tissues
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The cervical tissues were fixed in 4% PFA at 4 °C overnight, washed with PBS, and then embedded in paraffin. The tumour tissues were sliced into 10-μm-thick sections and dried overnight at 37 °C on glass slides. The dried slices were deparaffinized in xylene first followed by rehydration through a graded concentration of alcohol. Hydrogen peroxide (3%) was used to quench the sections, and 5% bovine serum albumin (BSA) was added to block the slices for 1 h at room temperature. Primary antibody (anti-Cdc25A; 1:500; ThermoFisher, #PA5-77092, USA) was added and incubated with the slices at 4 °C overnight. The next day, the antibody was washed off with PBS, and secondary antibodies were added and incubated with sections for 2 h at room temperature. Substrates of the Envision system-HRP from the kit (Dako REAL Ebision kit; DAKO, Glostrup, Denmark) were added to incubate with stained slices as the manufacturer’s protocol described, and the signals were analysed with a light microscope.
5
IHC Staining of FFPE Tumor Tissues
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Formalin (10%)-fixed and paraffin-embedded (FFPE) tumour tissue samples were prepared. The FFPE sample were cut into 5 μm sections. For IHC staining, sections were first treated with BSA (5%) at room temperature for 1 hour. Blocked sections were then treated with CD31 primary antibody (#77,699, Cell signalling Technology, USA) at 4 °C on a shaker overnight. Then, after washing with PBS twice, the sections were incubated at room temperature for 2 h with secondary antibodies. Sections were washed again and incubated with HRP (Dako REAL Ebision kit; DAKO, Denmark) according to the manufacturer's instructions.
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