Creative Biolabs has accumulated extensive experiences in IHC-Positive Hybridoma Screening. For antibodies intended for immunohistochemical (or immunocytochemical) staining, in addition to the native immunogens, e.g. recombinant protein, we may alternatively use denatured antigens in animal immunization and hybridoma screening. Our theory is that antigens to be stained in IHC are denatured (completely or partially), altered (by cross-linking) or precipitated by paraffin embedding and various fixations. We may use fixative-treated immunogens in animal immunization. In the end, we perform hybridoma screening with IHC using paraffin-embedded slides or tissue microarrays at extra charges. Customer may provide the slides or tissue arrays; otherwise, we may provide the slides or arrays at customers' expense.
In IHC, it is not easy to know the specificity of the antibodies, since a perfect control is usually not available, except in the cases in which there is a gene-knock out tissue to be used as a control or a specific transgenic fusion protein can serve as a reference staining. For this reason, an independent assay, such as ELISA, Western Blotting or IP is required to confirm the specificity of the antibody's binding ability to the target antigen. After that, the antibody will be tested in IHC assays and the staining must be specific, i.e. positive not to all tissues or cells and the staining is stronger than the control isotype-matched negative control antibodies. For this reason, to get IHC-positive antibodies, we first use ELISA, Western Blotting (for denatured epitopes) and/or Immuno-precipitation (for native epitopes) to select the positive binders. In practice, we found that if we obtain many ELISA/Western/IP positive clones, the chance for us to have an IHC positive clone is very big. It is important to conduct ELISA, Western Blotting and/or Immuno-precipitation to validate the specificity of the IHC-positive antibodies, although this strategy is widely-forgotten.
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