Enhanced Research Applications for IHC
Michael Gandy and Craig Barker, Leica Microsystems
A wide variety of histological techniques have long been employed in cancer research and diagnostics. Historically these techniques, such as immunohistochemistry (IHC) and in situ hybridisation (ISH), have been manual, time consuming and labour intensive. In the field of histology, three major technical advancements over the last decade – High volume tissue micro-arraying, Fully automated IHC and ISH, and slide scanning and image analysis – have enabled histological investigation to remain as one of the key life science research tools used today.
Multiple chromogenic labelling
Yet to find widespread application in the diagnostic setting, multiple labelling is a technique that has been successfully performed in various formats for a number of years. Two of the main uses of multiple labelling techniques have been their utilisation in evaluating antigen versus antigen or antigen versus gene expression in samples with limited viable cellular material or in samples where cellular differentiation and/or antigenic expression in adjacent cellular targets require same slide evaluation. Figure 1 illustrates a case of Hodgkin’s Lymphoma stained with Novocastra™ antibodies to CD3 (clone LN10), CD20 (clone MJ1) and CD30 (clone 1G12).
Direct immunofluorescence
The application of direct immunofluorescence is a useful research tool for assessing primary antibody specificity. The lack of subsequently applied chromogenic detection chemistry allows the assignment of a specific fluorescence signal to the antibody under evaluation, ruling out any potential detection system cross reactivity. This technique can often be used with multiple marker/multiple fluorophore labels to demonstrate and differentiate a range of highly specific cellular components. Figure 2 illustrates fully automated direct immunofluorescence of the SKBR-3 human breast cancer cell line stained with the anti-FITC conjugated mouse monoclonal anti-HER2 antibody clone CB11 and counterstained with DAPI. Note the slight granular appearance of the direct fluorescent signal attributed to the specific heterogeneity of membrane HER2 expression. This specific membrane expression pattern is often lost when layered chromogenic signal amplification is applied.
Cytological immunopreparations
The performance of immunocytochemistry on cytological preparations is again commonplace in the clinical setting, but it also has a role within cancer research. The use of cytological preparations in tumour biology research allows for cellular antigens to be evaluated and localisation determined without the artifacts induced by the rigours of prolonged formalin fixation and paraffin processing. Figure 3 shows dual colour labelling on an in-house grown, polyethylene glycol (PEG) fixed, mixed cell-line cytospin of Ramos (B-cell Burkitts lymphoma) and Jurkat (T-cell leukaemia) with Novocastra antibodies to CD3 (clone LN10) and CD20 (clone MJ1), with a light hematoxylin counterstain. Note the polar membrane localisation of CD20 together with blanket membrane and cytoplasmic staining of the CD3 antigen.
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- Fig. 1: Hodgkins Lymphoma stained with Novocastra antibodies to CD3 (clone LN10), CD20 (clone MJ1) and CD30 (clone 1G12)
- Fig. 2: SKBR-3 (3+) Human Breast Cancer Cell Line stained with HER2 (CB11)-FITC + DAPI counterstain
- Fig. 3: Mixed cell-line cytospin of Ramos (B-cell Burkitts lymphoma) and Jurkat (T-cell leukemia) with Novocastra antibodies to CD3 (clone LN10) and CD20 (clone MJ1) with light hematoxylin counterstain
- Note: All techniques have been stained using the fully automated Leica Bond IHC and ISH system with Research Platform enabled, and have not been validated for clinical use.