Western Blot as a Support Technique for Immunohistochemistry to Detect Programmed Cell Death Ligand 1 Expression

Parra, Edwin R.; Ruiz, Sharia Hernández

doi:10.1007/978-1-0716-1278-1_5

Cited by 1 publication

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“…This strategy can also be used to ensure that any antibody validation performed in-house uses the most relevant biological models for the targets of interest. Although immunostaining techniques that are established in a lab, such as Western blot ( Parra et al, 2018b) , in positive and negative cell lines ( Bordeaux et al, 2010) for research antibodies, can help provide a quick visual indication of antibody specificity ( Parra et al, 2020 ; Parra and Hernández Ruiz, 2021b) , it is always important and recommended that the antibody’s data generated be supported by orthogonal testing. One way of achieving this is to mine publicly available databases (e.g., CCLE, BioGPS, Human Protein Atlas, DepMap Portal, COSMIC) for genomic and transcriptomic profiling information to clarify whether observed immunostaining results are relevant or are instead due to antibody-related artifacts ( Cell Signaling, 2019 ; Ghandi et al, 2019 ; Broad Institute, 2021 ; COSMIC, 2021 ; The Human Protein Atlas, 2021 ).…”

Section: Strategies For Antibody Validationmentioning

confidence: 99%

Best Practices for Technical Reproducibility Assessment of Multiplex Immunofluorescence

Fernández

Hernandez-Ruiz

Rojas

et al. 2021

Front. Mol. Biosci.

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Multiplex immunofluorescence (mIF) tyramide signal amplification is a new and useful tool for the study of cancer that combines the staining of multiple markers in a single slide. Several technical requirements are important to performing high-quality staining and analysis and to obtaining high internal and external reproducibility of the results. This review manuscript aimed to describe the mIF panel workflow and discuss the challenges and solutions for ensuring that mIF panels have the highest reproducibility possible. Although this platform has shown high flexibility in cancer studies, it presents several challenges in pre-analytic, analytic, and post-analytic evaluation, as well as with external comparisons. Adequate antibody selection, antibody optimization and validation, panel design, staining optimization and validation, analysis strategies, and correct data generation are important for reproducibility and to minimize or identify possible issues during the mIF staining process that sometimes are not completely under our control, such as the tissue fixation process, storage, and cutting procedures.

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