Even the most seasoned antibody researchers would attest that generating antibodies for immunohistochemistry and immunocytochemistry applications can introduce a special set of challenges.
In Intracellular cytokine (IC) staining, for example, you have to fix and permeabilize cells. Then there are the intricacies involved in recognizing an intracellular target when trying to generate an antigen: You’ve got to design the right peptide that will hit the intended target when it should. And as with any other antibody development initiative, “speed” and “efficiency” remain inescapable project drivers.
That’s why we’ve invested considerable energy and resources toward building an in-house expertise and infrastructure in immunohistochemistry and immunocytochemistry (including high-throughput flow cytometers that can sort 6,000 to 8,000 clones each week). Here are some examples of how we’ve put our skills and resources to work on behalf of Antibody Solutions clients:
- Immunohistochemistry and Immunocytochemistry
We produced a series of antibodies to human cyclooxygenase 2 (COX-2), one of which, AS66-P, is particularly useful for immunohistochemistry applications. COX-2 is a proinflammatory enzyme that is induced at sites of inflammation and dysregulated in many cancers. Our antibody was used for analysis of COX-2 expression in cystectomy biopsies of bladder cancer patients during a five-year follow-up study. In this study, Liu et al (2001) used A66-P immunostaining of paraffin-embedded bladder tissue and showed that COX-2 expression in bladder tumor biopsies correlated with survival. Using a custom-designed monoclonal antibody manufactured by our team against the Drosophila melanogaster homologue of p53, Ollmann et al. (Cell, 101:91, 2000) revealed an ancestral pro-apoptotic function for p53 and identified D. melanogaster as an ideal model system for elucidating the p53 apoptotic pathways induced by DNA damage.
- Intracellular Staining
Antibody Solutions has a long history of developing antibodies suitable for intracellular staining techniques. Our high-throughput flow cytometry screening platform, for example, has proven to be especially beneficial for more rapid discovery of antibodies to intracellular proteins. We’re familiar with various fixation/permeabilization methods to use when evaluating antibody candidates, and offer a range of cytokine-specific antibodies for intracellular flow cytometry applications, including AS5-F (human IL1-α), AS10-F ( IL1-β), AS12-F ( IL-6) and AS13-F (IL-8).
Intracellular Cytokine Staining
Intracellular cytokine staining circumvents the need to isolate populations of cells by physical means such as panning methods, columns, density gradients, etc., which rarely yield populations of cells of sufficient purity that allow for easy data interpretation. So our team conducted intracellular staining of peripheral blood mononuclear cells with our carboxyfluorescin-conjugated monoclonal antibody AS1-F to human tumor necrosis factor alpha (TNFα). This cytokine is secreted from many leukocytes in response to stimulation with bacterial lipopolysaccharide (LPS). As shown in the results below, when stimulated with LPS and Phorbol 12-Myristate-13-Acetate (PMA), only the CD16-positive cell population (mostly neutrophils) but not the CD4+ T-helper cell population responds with TNFÎ± synthesis (in the left panel of the diagram, PBMC are gated into CD4+, CD16+ and other cells).
Simple truth is, adding expertise in immunohistochemistry and immunocytochemistry to your next research project can help eliminate some common frustrations and slowdowns traditionally associated with antibody generation. We’d welcome the opportunity to learn more about your specific goals and needs, so contact us to get started today.