Staining was performed on 4μm thick FFPE sections by using automated staining. After deparaffinization, slides were treated with antigen retrieval (AR) buffer (Diva Decloaker from BioCare Medical or Leica Bond Epitope Retrieval Solution 2) and heated for 15 mins at 95–100°C. Slides were allowed to cool in the AR buffer for 15 min at room temperature and were then rinsed with deionized water and 1 × Tris-buffered saline with Tween-20. Endogenous peroxidase was blocked using 3% hydrogen peroxide. Protein stabilization and background reduction was done using intelliPATH™ Background Punisher. Slides were then incubated for 1h with primary antibodies against HLA-ABC (clone EMR8–5), PD-L1 (clone RBT-PDL1) followed by the secondary antibody (PerkinElmer OPAL Polymber HRP Ms Plus Rb) application for 30 minutes and the application of the tertiary TSA-amplification reagent (PerkinElmer OPAL fluor) for 10 minutes. Antigen stripping was performed either by heating with Leica Bond Epitope Retrieval Solution 2 or with Biocare medical denaturation reagent at room temperature. Slides were imaged with either Leica Aperio FL Immunofluorescence slide scanner or Leica SP8 confocal microscope.
Opal polymber hrp ms plus rb
Manufactured by PerkinElmer
The OPAL Polymber HRP Ms Plus Rb is a laboratory equipment product from PerkinElmer. It is designed to perform immunohistochemistry (IHC) analysis. The product enables the visualization of target proteins in tissue samples using a horseradish peroxidase (HRP) enzyme-based detection system.
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2 protocols using opal polymber hrp ms plus rb
1
Immunofluorescence Staining of FFPE Tissue
2
Automated Immunohistochemical Staining Protocol
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Staining was performed on 4-μm-thick formalin-fixed paraffin-embedded (FFPE) sections by using automated staining. After deparaffinization, slides were treated with antigen retrieval (AR) buffer (Diva Decloaker from BioCare Medical or Leica Bond Epitope Retrieval Solution 2) and heated for 15 minutes at 95°C–100°C. Slides were cooled in the AR buffer for 15 minutes at room temperature and then rinsed with deionized water and 1 × TBS with Tween-20. Endogenous peroxidase was blocked using 3% hydrogen peroxide. Protein stabilization and background reduction was done using intelliPATH Background Punisher. Slides were then incubated for 1 hour with primary antibodies against B7-H3 (clone RBT-B7H3) followed by the secondary antibody (PerkinElmer OPAL Polymber HRP Ms Plus Rb) application for 30 minutes. Staining was done either by DAB (3, 3′-diaminobenzidine) or fluorochrome. Tertiary TSA-amplification reagent (PerkinElmer OPAL fluor) for 10 minutes. Antigen stripping was performed by heating with Biocare medical denaturation reagent at room temperature. Slides were imaged with Leica SP8 confocal microscope or scanned by Aperio Scanscope.
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