The VENTANA PD-L1 (SP142) rabbit monoclonal primary antibody (Ventana Medical Systems Inc.) was optimized for use as a fully automated IHC assay on the BenchMark ULTRA (Ventana Medical Systems Inc., Tucson, AZ) staining platform using the OptiView DAB IHC Detection Kit and OptiView Amplification Kit (Ventana Medical Systems Inc., Tucson, AZ). The assay was optimized for detection of PD-L1 expression in NSCLC, where TC and IC are predictive8 (link) and in UC, where IC is predictive.11 (link) Parameters evaluated during optimization included antibody concentration, antibody diluent, multiple antigen retrieval methods, antibody incubation conditions, and counterstain conditions. In addition, signal amplification using the OptiView Amplification Kit (Ventana Medical Systems Inc.) was tested to evaluate its efficacy in visualizing IC staining. The optimal conditions for staining both TC and IC in NSCLC and UC tissues on the BenchMark ULTRA instrument are outlined in Table 1 . Briefly, antigen retrieval was undertaken for 48 minutes, the primary antibody was applied for 16 minutes at 36°C, amplification was done for 8 minutes amplifier/8 minutes multimer, and samples were counterstained for 4 minutes with hematoxylin II and postcounterstained for 4 minutes.
Ventana pd l1 sp142 rabbit monoclonal primary antibody
Manufactured by Roche
Sourced in United States
The VENTANA PD-L1 (SP142) rabbit monoclonal primary antibody is a laboratory instrument used for the detection of PD-L1 protein expression in tissue samples. This antibody is designed for use with specific VENTANA automated staining systems.
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3 protocols using ventana pd l1 sp142 rabbit monoclonal primary antibody
1
Automated IHC Assay for PD-L1 Detection
2
Automated PD-L1 Immunohistochemistry Assay
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PD-L1 on TIICs was measured using the VENTANA PD-L1 (SP142) rabbit monoclonal primary antibody (Ventana Medical Systems, Tucson, USA) with a fully automated IHC assay on the BenchMark ULTRA (Ventana Medical Systems, Tucson, USA) staining platform according to manufacturer protocols. The assay was optimized for the detection of PD-L1 in urothelial carcinoma, for which TIICs are predictive. The VENTANA PD-L1 (SP142) stain highlights a heterogeneous population of immune cells including lymphocytes, macrophages, dendritic cells, and granulocytes. In our study, most immune cells are blood-origin lymphocytes, and some granulocytes have been identified.
Briefly, formalin-fixed, paraffin-embedded tissue sections were cut in widths of 1.5 μm. After deparaffinization, antigen retrieval was performed using cell conditioning reagent 1 (Ventana Medical Systems, Tucson, USA). After primary antibody incubation at 37°C for 32 minutes, the Ultra View DAB Detection Kit (Ventana Medical Systems, Tucson, USA) was used for visualization. The slides were washed in distilled water, counterstained with hematoxylin (12 minutes) and bluing reagent (4 minutes), dehydrated in a descending order of alcohols, cleared in xylene, and coverslipped with Tissue-Tek mounting medium (Sakura Finetek Japan, Tokyo, Japan).
Briefly, formalin-fixed, paraffin-embedded tissue sections were cut in widths of 1.5 μm. After deparaffinization, antigen retrieval was performed using cell conditioning reagent 1 (Ventana Medical Systems, Tucson, USA). After primary antibody incubation at 37°C for 32 minutes, the Ultra View DAB Detection Kit (Ventana Medical Systems, Tucson, USA) was used for visualization. The slides were washed in distilled water, counterstained with hematoxylin (12 minutes) and bluing reagent (4 minutes), dehydrated in a descending order of alcohols, cleared in xylene, and coverslipped with Tissue-Tek mounting medium (Sakura Finetek Japan, Tokyo, Japan).
3
Automated IHC Assay for PD-L1 Expression in Urothelial Carcinoma
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The VENTANA PD-L1 (SP142) rabbit monoclonal primary antibody (Ventana Medical Systems, Tucson, AZ, USA) was used as a fully automated IHC assay on the BenchMark ULTRA (Ventana Medical Systems) staining platform to measure PD-L1 expression on tumor-infiltrating ICs following routine protocols and specific manufacturer instructions. This assay was optimized for the detection of PD-L1 expression in urothelial carcinoma, where IC is predictive.20 (link) The results were analyzed by a pathologist specialized in genitourinary cancer who was blinded to the clinicopathologic and survival data. PD-L1 expression level was categorized into the following three groups based on the percentage of the tumor area covered by PD-L1 expression on ICs: IC0 (<1%), IC1 (≥1% and <5%), and IC2/3 (≥5%).21 (link),22 (link) In addition, PD-L1 expression was dichotomized as positive (≥5%) or negative (<5%) using a 5% cutoff value. Representative images of PD-L1 expression on ICs are shown in Figure 2 .![]()
Representative images showing immunohistochemical staining of programmed death ligand-1 on tumor-infiltrating immune cells in radical cystectomy specimens. (
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