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Antibody Validation By Immunocytochemistry


IMPORTANT: Use the same fixation and staining conditions that will be used for ICW Assays!


Figure 1. PARP-1 (cleaved) antibody specificity in apoptotic cells. Immunocytochemical analysis of HeLa cells, either untreated or treated with 1 μM Staurosporine for 4 h to induce apoptosis. Mitosciences PARP-1 (cleaved) In-Cell ELISA Kit (IR) (ab110215) was used. The PARP-1 (cleaved) antibody stains cell nuclei, but only after STS treatment.

Fluorescent signals can be assessed by near-infrared fluorescence microscopy, or microplate assays imaged with the Odyssey® Classic, CLx, or Sa Imagers.

Guidelines for validation:

  • Choose a primary antibody that is validated for immunofluorescence (IF)1
    • Antibody must recognize fixed protein epitopes
  • Confirm cellular localization of target proteins by immunofluorescent microscopy1 (Fig. 1)
  • Confirm up- or down-regulation of target protein expression under various conditions (Fig. 1)
  • Use a blocking peptide to pre-adbsorb the primary antibody and reduce specific signal
  • Titrate antibodies for optimal signal and lowest background

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

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