Immunohistochemistry (IHC) Services

XpressWay^TM Profiles and CustomMapping^TM provide a high level assessment of the expression patterns of genes in human tissue. However, it is also important to identify the pattern of expression of a target protein at the cellular level in order to gain a greater understanding of the potential function of that protein and its possible role in disease.

Asterand has been providing high quality IHC data to our customers for over 10 years. Our extensive human tissue biorepository allows us immediate access to high quality human tissues representing a range of therapeutic areas, enabling us to generate IHC data quickly. We have experience in providing IHC services to support oncology, respiratory, inflammation and metabolic disease research.

Stage 1: Assay optimization / validation

• FFPE or frozen tissue sections
• Positive and negative control tissues
• 1 to 3 antigen retrieval methodologies (FFPE) or 2 fixation techniques tested (frozen)
• 3 to 5 primary antibody concentrations with matching non-immune IgG isotype and 'no primary antibody' controls
• Positive control antibody, e.g. von Willebrand's Factor or pancytokeratin, included with every assay to validate detection reagents
• Standard Stage 1 report defining and recommending optimal IHC conditions

Typical optimization of IHC assay parameters using 3 antigen retrieval techniques, 3 primary antibody concentrations and corresponding non-immune IgG controls. This example shows optimization of conditions for pro-insulin localization in human pancreas.

Stage 2: Assay

• Validated IHC protocol with matching non-immune IgG isotype and 'no primary antibody' controls
• Number of donors and tissue types chosen by the customer
• Flexible reporting options are available:
  • representative region-matched images of primary antibody with IgG isotype controls
  • description of immunoreactive tissue structures
  • analysis of pattern and intensity of target immunoreactivity
  • discussion and conclusions
• Assay slides can be provided for review and analysis
• If cellular phenotyping data are required, this may be incorporated directly into this experimental stage or may be added through a follow-up study

Her-2 testing in breast cancer tissue samples.

Cellular phenotyping
Identification of specific cell populations that express a target of interest can be achieved using dual labeling immunofluorescence. This can provide further detail and confirm the specific phenotypes of target immunoreactive cells.

Asterand is experienced in dual labeling techniques using both tissue sections and isolated primary cells. Using an immunofluorescence approach with a panel of characterized antibodies (including CD3, CD4, CD8, CD15, CD20cy, CD56, CD68, mast cell tryptase), Asterand has characterized target expression in a range of specific inflammatory cell types in lung parenchyma sections. Examples of data generated are shown below.

GPCR expression in human neutrophils (CD15) and human alveolar macrophages (CD68) present in human lung parenchyma sections, visualised by dual label immunofluorescence.

Human Tissue Microarrays
The throughput of IHC studies can be vastly increased with the use of tissue microarrays (TMAs). Asterand can construct custom made FFPE or frozen human TMAs and have two standard TMA formats for IHC:

• Cross Reactivity Assay (frozen, human non-diseased TMA designed to represent the FDA recommended human tissue panel for therapeutic antibody cross reactivity studies)
• XpressArray^TM (FFPE, human non-diseased TMA designed to complement XpressWay^TM gene expression profiles

To download details of Asterand's TMA approaches for target localization, click here.

For further information, contact us at advantage@asterand.com