Figure 2

Schematic representation of a suggested approach to adopt in the optimisation of 'research-only' antibodies. To generate ideal controls for the optimisation of antibodies for immunohistochemical analysis of formalin-fixed, paraffin-embedded tissues, we propose the use of optimal controls in the form of cell lines. First, cell lines that overexpress the gene and protein of interest are identified by mining publicly available databases (for example, microarrays and proteomics). The expression of the gene and protein in the cell lines identified as 'overexpressors' is validated by Western blot analysis and quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR) assay. These validated cell lines are used as positive controls. Once this validation step is completed, the gene of interest is silenced using validated short interfering RNA (siRNA}. The process of gene silencing is subsequently validated by qRT-PCR assay and Western blot analysis, and these cells are used as negative controls. After in vitro validation, pellets of the positive and negative control cell lines are produced and then subjected to formalin fixation and paraffin embedding using routine methods. These controls are then used for optimisation of the antibody titration and choice of antigen retrieval system. Please note that the siRNA negative control has an internal positive control, given that the efficiency of siRNA silencing almost never reaches 100%. In addition, multi-tumour blocks and tissue microarrays constructed with distinct types of tissue can be used as positive and negative controls (images of routine diagnostic slides courtesy of CM). CTRL: control; FFPE: formalin-fixed paraffin-embedded; IHC: immunohistochemistry; siRNA: short interfering RNA; TMA: tissue microarray; WB: Western blot analysis.