Autoradiography as a Simple and Powerful Method for Visualization and Characterization of Pharmacological Targets

Autoradiography is a powerful and simple technique to study distribution of protein binding sites in tissues using specific radioligand for the target. It's also suitable alternative to immunohistochemistry. This technique visualizes protein expression with spatial resolution via digital images which is faster than traditional film exposure.

It may also be used for pharmacological analysis of protein targets. The method involves mouse tissues mounted on glass but may be extended to other species or even to cells grown on coverslips. Demonstrating the procedure will be by Nane Griem-Krey, a PhD student in my laboratory.

To begin, submerge the tissue in powdered dry ice to snap freeze it. Transfer the frozen tissue directly to a cryostat that is set to minus 20 degrees Celsius. Let the tissue acclimate to minus 20 degrees Celsius in the cryostat for 20 minutes.

Then cover the tissue holder with embedding medium outside the cryostat and quickly place the frozen tissue specimen in the desired orientation while the embedding medium is still liquid. Transfer the tissue holder back to the cryostat and expose the embedding medium to temperatures below minus 10 degrees Celsius for hardening. Position the tissue holder in the microtome of the cryostat.

Adjust the orientation of the tissue to avoid sloped sections. Cut the tissue with the guidance of a stereotaxic atlas in sections of desired thickness and unfold the section with a small brush if necessary. Thaw mount the section onto a microscope slide.

Then sequentially collect the sections from the region of interest for the desired technical repetition. Allow the sections on the slides to air dry for one hour before further handling. In a certified radioactivity laboratory, use a pencil to label the slides with the experimental conditions.

Place the slides horizontally in plastic trays. Carefully apply an appropriate volume of SA buffer adjusted to the target in question to the sections mounted on the slides. Make sure that each section is completely covered.

For the determination of non-specific binding, supplement SA buffer with a relevant concentration of unlabeled compound. Then cover the trays with lids to avoid evaporation. Pre-incubate at a relevant temperature for 30 minutes on a plate shaker set to 20 rpm.

Next, pour off the pre-incubation liquid from each slide and transfer the slides back into the plastic tray. Immediately cover the sections completely with the relevant concentration of radioligand in SA buffer. For the determination of non-specific binding, supplement radioligand solution with a relevant concentration of unlabeled compound.

Cover the trays with lids and incubate under the desired conditions with constant gentle shaking at 20 rpm. After this, pour off the incubation solution and transfer the slides into a microscope slide rack. Immediately wash the slides.

For the GHB protocol, wash twice with ice cold SA buffer for 20 seconds and then rinse twice by dipping the slide rack into trays filled with ice cold distilled water. Position the slides vertically in racks and air dry for at least one hour. Then transfer the slides to a fixator containing paraformaldehyde for overnight fixation at room temperature.

The next day, transfer the slides to a desiccator box containing silica gel at room temperature. Leave the slides in the desiccator box for three hours to eliminate moisture. First, place the sections into a radiation shielded imaging plate cassette with the tissue facing up.

Include a radioactive microscale in every cassette for subsequent quantification of radioligand binding. Immediately before usage, load the tritium-sensitive phosphor imaging plate into the phosphor imaging machine and expose it to visible infrared light according to the manufacturer's instructions to erase it. Then remove the imaging plate from the phosphor imaging machine and immediately place it onto the sections in the cassette.

Make sure that the cassette is closed completely. Expose the sections to the phosphor imaging plate for the optimized exposure time at room temperature while shielded from light. After exposure, carefully open the cassette in the dark and immediately transfer the imaging plate into the dark box of a phosphor imager.

Scan the plate at the highest resolution possible to obtain a digital image. To begin, open an appropriate image analysis program. Determine the relative optical densities for each calibration standard by selecting the menu item region determination.

Select the tool for region creation and use it to select an area of equal size for each point of the radioactive microscale. Assign a number to each selected area by clicking on number under the menu item label. Click File Export and then 2D Region Report to export the OD values for each point of the calibration standard.

In the proprietary imaging software, use a region creation tool to select the region of interest in every section and measure its ODs to begin performing quantification of the autoradiograms. Select the same region in every section by creating a template for the region of interest and copy and adjust it to minor variations in brain anatomy in each autoradiogram. After this, click File Export 2D Region Report to export the ROD values and sizes of selected areas into a spreadsheet.

Determine the binding of the radioligand as outlined in the text protocol. In this study, the anatomical distribution of the high affinity GHB binding sites are visualized with the radiolabeled GHB analog radioactive HOCPCA in a mouse brain which was cut into coronal, sagittal, and horizontal sections. High levels of binding are seen in the hippocampus and cortex while lower binding levels are seen in the striatum and no binding is detected in the cerebellum.

As shown here, the anatomical structures may be visualized using different planes and anatomical integrity may be supported by cresyl violet staining. Representative autoradiograms of a rat, a mouse and a pig illustrate the evolutionary conservation of the high affinity GHB binding sites in the mammalian brain. Radioactive HOCPCA binding sites are detected in all three species enabling comparison of gross brain anatomy between them.

The high affinity GHB binding sites are probed with GHB radioligands which display different affinities for the binding sites but comparable specific activities. Radioactive HOCPCA is endowed with a high sensitivity and excellent signal to noise ratio. Due to the lower sensitivity of radioactive NCS-382, higher radioligand concentrations must be used in order to obtain similar levels of binding.

Even higher radioligand concentrations are necessary for radioactive GHB to achieve comparable binding levels. However, higher radioligand concentrations also increase the level of non-specific binding with a consequently lower signal to noise ratio. It is important to optimize the assay conditions like the radioligand concentration, washing and exposure time in order to get the best signal to noise ratios and it's also very important to remember to erase the plate before.

The procedure can be extended to in vivo conditions to get an idea about compound binding in a living animal which could be useful for discovery projects. Remember to work in a laboratory with certified full radioactive material and to wear protective clothing when handling radioactivity or paraformaldehyde.