Receptor Autoradiography

We use Bright cryostat for precision sectioning of fresh-frozen tissue, enabling the thin, well-preserved sections essential for high-resolution receptor autoradiography.

What Is Receptor Autoradiography Used For?

In vitro autoradiography. Using sections cut from frozen untreated human or animal tissue, in vitro autoradiography measures the affinity of an unlabeled drug by competing with the binding of a radiolabeled drug to the target receptor. In vitro autoradiography can also identify off-target binding sites, by revealing residual binding in knockout animal tissue, or in regions where the target receptor is not expressed.

Ex vivo autoradiography, performed on tissue from drug-treated animals, determines the receptor occupancy of the drug at the time of tissue collection. Because the technique localizes binding to anatomically defined regions, it reveals heterogeneity that whole-tissue homogenate binding misses, such as differential occupancy between the different brain regions (e.g., cortex, hippocampus, cerebellum).

For human tissue use, ethics committee approval is required. Gifford Bioscience manages the application process to obtain the tissue from an accredited UK tissue bank.

Example Data

Binding of [³H]DAMGO to µ opioid in coronal sections at the level of the corpus striatum in a rat brain.

Binding of [³H]LRRK2-IN-1 to the LRRK2 enzyme in rat kidney. Sections in the centre and to the left are total binding. The two sections on the right are non-specific binding, defined using unlabeled LRRK2-IN-1 (10 µM).

When to Choose Autoradiography over Homogenate Radioligand Binding?

Homogenate radioligand binding is the right tool for affinity, density and competition measurements, averaged across a tissue. Receptor autoradiography is the right tool when spatial information matters, such as:

• regional receptor distribution mapping;
• identification of off-target binding sites;
• ex vivo receptor occupancy;
• work in low-abundance regions where homogenate dilution would lose signal;
• translational studies, where preserving anatomy is essential for read-across to in vivo imaging.
• The two approaches are often used in combination, with homogenate binding establishing affinity and autoradiography confirming spatial selectivity.

The two approaches are often used in combination, with homogenate binding establishing affinity and autoradiography confirming spatial selectivity.

Autoradiography Protocol

Cryostat-cut tissue sections are incubated with the radiolabelled drug until equilibrium is reached. The sections are washed in fresh buffer to remove unbound radiotracer, dried and imaged using quantitative phosphorimaging. Regions-of-interest (ROIs) are then drawn over each imaged section and radioactivity levels are measured. Even small regions, smaller than 100 μm, can be quantified.

Non-specific binding is defined on adjacent sections using an excess of unlabelled competitor. Specific binding is calculated by subtraction. Calibrated standards co-imaged on the same phosphor screen convert phosphorimage signal to fmol of bound radioligand per mg of tissue, allowing direct comparison across regions, tissues and studies.

The Gifford autoradiography workflow, from cryostat sectioning through radioligand incubation, washing, phosphorimaging and ROI-based quantification. The in vitro route maps receptor distribution and binding; the ex vivo route measures receptor occupancy in tissue from drug-treated animals.

Applications across Target Classes

Receptor autoradiography is target-class agnostic. Any binding site that can be labelled with a selective radioligand can be mapped and quantified, including GPCRs, ligand-gated and voltage-gated ion channels, transporters, enzymes, and aggregated proteins such as tau, amyloid-β and α-synuclein. The same workflow applies in each case, with the radioligand and incubation conditions tailored to the target.