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Keys to Optimizing the
In‑Cell Western Assay

  • Carefully select and validate primary antibodies
  • Use optimal fixation and staining conditions
    • Find the "best" fixative for your protein target
      • Options include formaldehyde, methanol/acetone, Prefer
    • Start with conditions that work for immunofluorescent staining of that target
    • Follow recommendations from the primary antibody vendor
    • Read more about fixation and permeabilization
  • Run appropriate controls
    • Wells with secondary antibody only (no primary antibody)
    • Conditions that up- or down-regulate expression of the target
    • Western blotting and immunofluorescent microscopy to confirm effects of treatment
    • Replicates, for increased precision
  • For more Optimization Tips, Go to FAQs & Tech Tips.

1. Aguilar HN et al. PLoS ONE 5(4): e9965 (2010)

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