Paraffin embedded tissue specimens were cut at 4 μm serial sections on positively charged slides, routinely processed, air-dried overnight at 37°C, deparaffinized in xylene and rehydrated in a graded alcohol sequence. The slides were incubated in 3% hydrogen peroxide for 5 min at RT to block endogenous peroxidase activity and incubated in blocking solution (CSA II Kit, Dako, Glostrup, Denmark) for 90 min at RT. Slides were incubated overnight with primary antibodies (Abnova 1:500). Slides were rinsed with TBST, bound antibody was detected using the CSA system embodies technology (Dako CSA II, Biotin-Free Catalyzed Amplification System, Glostrup, Denmark) and visualized by diaminobenzidine tetrahydrochloride. The samples were counterstained with hematoxylin. Finally, the samples were dehydrated in a graded alcohol sequence and mounted in Richard-Allan Scientific Cytoseal XYL (Thermo Scientific, Waltham, MA, USA).
Biotin free catalyzed amplification system
Manufactured by Agilent Technologies
Sourced in Denmark
The Biotin-Free Catalyzed Amplification System is a laboratory equipment product offered by Agilent Technologies. It is designed to perform nucleic acid amplification without the use of biotin. The core function of this system is to facilitate the amplification of specific DNA or RNA sequences in a biotin-free environment.
Automatically generated - may contain errors
2 protocols using biotin free catalyzed amplification system
1
Immunohistochemical Staining of Paraffin-Embedded Tissue
2
Standardized PD-L1 Immunohistochemistry Evaluation
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Cells were fixed in 95% ethanol and embedded in paraffin. Egg albumin was used for cell aggregation. For ICC and IHC, 4 μm thick sections from FFPE tissue blocks were cut using a microtome and routinely deparaffinized. The sections were incubated with 0.3% hydrogen peroxide to block endogenous peroxidase activity. Antigen retrieval was performed in 0.01 M of citrate buffer (pH 6.0) or Tris–EDTA buffer (10 mM Tris at pH 9.0, 1 mM EDTA, 0.03% Tween 20) at 95 °C. Three different PD-L1 antibodies (DAKO 22C3, 1:50; VENTANA SP142, 1:50; and VENTANA SP263, 1:50) were used for immunocytochemical and immunohistochemical staining. For SP142 IHC amplification and DAB development, the Biotin-Free Catalyzed Amplification System (DAKO, K1497) was used. For SP263 IHC amplification and DAB development, the OptiView DAB IHC Detection Kit (VENTANA, 760-700) was used according to the manufacturer’s instructions. Each slide was counterstained with hematoxylin and then mounted.
To evaluate the IHC results of tissue samples including both whole sections and TMAs, each case was separated into groups with < 1% (negative), 1–49% (low), or ≥ 50% (high) positive tumor cells. A tumor cell with membranous staining, at least weak and partial, counted as a positive tumor cell.
To evaluate the IHC results of tissue samples including both whole sections and TMAs, each case was separated into groups with < 1% (negative), 1–49% (low), or ≥ 50% (high) positive tumor cells. A tumor cell with membranous staining, at least weak and partial, counted as a positive tumor cell.
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