Molecular Imaging...One Probe Simplifies Discovery

Workflow

Target Identification: in vivo imaging projects typically begin with identification of a possible tracking agent or probe, such as a ligand to a receptor or specific antibody.

Label/Conjugation: The probe of choice can be labeled via NHS ester conjugation with IRDye® 800CW infrared dye for imaging on the Odyssey System. Protein labeling kits are available that use IRDye® 800CW NHS ester to label antibodies and proteins. Alternatively, IRDye® 800CW EGF is a labeled targeting agent for use in common human cancers that over-express EGFR.

In vitro Assays: Before starting a costly and time consuming in vivo experiment, probe specificity can be determined in vitro on the Odyssey System using an immunocytochemical assay performed in microplate format such as the cost effective In-Cell Western (ICW) assay.

Clearance and Characterization Studies in vivo: The labeled targeting agent should be evaluated in “tumor-negative” and “tumor positive” animals. Clearance studies address
important questions such as:

1. Is the probe retained beyond the intended targets (e.g. liver, kidneys, or bladder)?
2. How long does it take for the targeting agent to clear the animal?
3. What is the ideal probe concentration?
4. When is the best time post-injection to achieve the best signal-to-noise ratio?

Once probe clearance is determined, a full in vivo experiment can be performed with greater confidence. Up to three mice can be scanned and the IRDye-labeled probes visualized using the Odyssey System and MousePOD accessory.

Organs: More focused analysis of the probe’s localization can be done on the Odyssey by excising and scanning the target organ(s). Scans of organs provide a semi-quantitative estimate of signal and quickly show whether the targeting agent is present.

Tissue Sections: To determine exact tissue localization, frozen or paraffin embedded tissue sections can be scanned on the Odyssey at high resolution. Tissue sections can also be examined using a microscope equipped with an infrared filter.