The work was supported by the Lundbeck Foundation (Grant R133-A12270) and the Novo Nordisk Foundation (Grant NNF0C0028664). The authors thank Dr. Ales&#780; Marek for the supply of [3H]radioligand.

All animal handling was performed in compliance with the guidelines from The Danish Animal Experimentation Inspectorate.
NOTE: The protocol described here covers tissue preparation (i.e., mouse brain tissue), the in vitro autoradiographic assay in sufficient detail for setting up the method in a new lab, the exposure to phosphor imaging plates as well as subsequent densitometric analysis of autoradiograms (Figure 2) with the aim of localizing and quantifying radioligand binding in distinct anatomical structures. For histological comparison, a protocol for cresyl violet staining is included. Moreover, the determination of non-specific binding with a competing ligand is included within the protocol. For a detailed description on how to determine Kd, Bmax or Ki, see previous publication4.
1. Tissue Preparation by Cryosectioning
<ol>
	<li>Euthanize the mouse by cervical dislocation and immediately dissect out the brain using scissors and forceps. Directly proceed to the next step to avoid tissue damage.</li>
	<li>Snap-freeze the tissue by submersion in powdered dry ice, gaseous CO2 or isopentane. Directly transfer the frozen tissue to a cryostat with the temperature set to -20 &#176;C. Alternatively, store the tissue at -80 &#176;C until processing. 
	NOTE: Avoid repeated thawing/freezing to reduce tissue damage.</li>
	<li>Let the tissue acclimate to -20 &#176;C in the cryostat for 20 min before further processing to avoid tissue shattering.</li>
	<li>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. For instance, place the mouse brain vertically onto cerebellum in order to achieve rostral coronal sections. Transfer the tissue holder back to the cryostat and expose the embedding medium to temperatures below -10 &#176;C for hardening. 
	NOTE: Fragile tissue specimen should be coated in embedding medium within the tissue molds prior to mounting.</li>
	<li>Position the tissue holder in the microtome of the cryostat. Adjust the orientation of the tissue to avoid sloped sections.</li>
	<li>Cut the tissue with the guidance of a stereotaxic atlas18 in sections of desired thickness (12 &#181;m recommended for [3H] labelled ligands). Carefully straighten and unfold the section with a brush of small size if necessary and thaw-mount the section onto a microscope slide. Sequentially collect the sections from the region of interest for desired technical replication (e.g., 4 sections per slide).</li>
	<li>Allow the sections on the slides to air-dry for 1 h before further handling. 
	NOTE: Addition of desiccant material to slide boxes minimizes moisture build up on the tissue sections. Procotol can be paused here by storing sections long-term in slide boxes at -80 &#176;C.</li>
</ol>
2. In vitro Autoradiography
CAUTION: Radioactivity. Work in a certified laboratory according to local regulations. Wear protective clothing. Dispose in accordance with radioactive decay or outsource to a certified company.
<ol>
	<li>Thaw the sections for at least 30 min at room temperature (RT). Label the slides with experimental conditions. Use a pencil because the slides will be bathed in ethanol during subsequent staining.</li>
	<li>Place the slides horizontally in plastic trays. 
	NOTE: Positioning slides on an elevated platform within plastic trays facilitates their handling.</li>
	<li>Pre-incubate the sections mounted on the slides in assay buffer adjusted to target in question (for GHB protocol, 50 mM KHPO4 buffer pH 6.0 is used) by carefully applying an appropriate volume onto the slide (700 &#181;L for 3-4 rodent coronal sections). 
	NOTE: Make sure that every section is covered completely with liquid.
	<ol>
		<li>Cover the plastic trays with a lid in order to avoid evaporation and pre-incubate at relevant temperature (for GHB protocol pre-incubate for 30 min at RT) under constant gentle (20 rpm) shaking on a plate shaker.</li>
		<li>For the determination of non-specific binding, supplement assay buffer with relevant concentration of unlabelled compound (for GHB protocol, 1 mM GHB). 
		NOTE: Pre-incubation may not be necessary.</li>
	</ol>
	</li>
	<li>Pour off pre-incubation liquid from each slide and transfer the slides back to the plastic tray.</li>
	<li>To avoid dehydration, immediately incubate the sections with relevant concentration of radioligand in assay buffer under desired conditions (for GHB protocol, 1 nM [3H]HOCPCA for 1 h at RT) by covering the sections completely with the radioligand solution (700 &#181;L for 3-4 rodent coronal sections).
	<ol>
		<li>Incubate under under constant gentle (20 rpm) shaking of plastic trays with closed lid. 
		NOTE: The radioligand concentration can be validated by counting an aliquot in a liquid scintillation counter.</li>
	</ol>
	</li>
	<li>Remove the incubation solution by pouring off the liquid and transfer the slides into a microscope slide rack. Immediately proceed to the next step to avoid section dehydration.</li>
	<li>Wash the slides. For the GHB protocol, wash with ice-cold assay buffer twice for 20 s and then rinse twice by dipping the slide rack into the trays filled with ice-cold distilled water to remove salts. Position the slides vertically in racks for air-drying for at least 1 h at RT or dry the slides for 5 min with a blower set to cold temperature. 
	NOTE: Washing must be optimized, e.g., extensive washing may be useful for decreasing non-specific binding.</li>
	<li>Transfer the slides to a fixator containing paraformaldehyde (PFA) powder for overnight fixation with PFA vapours at RT in order to protect the integrity of the ligand-target complex. 
	CAUTION: PFA is toxic. Positionthe fixator in fume hood and avoid skin/eye contact with PFA.</li>
	<li>The following day, transfer the slides to a desiccator box containing silica gel for 3 h at RT to eliminate moisture.</li>
</ol>
3. Exposure to Phosphor Imaging Plates and Scanning
<ol>
	<li>Place the sections in a radiation-shielded imaging plate cassette with the tissue facing up. For subsequent quantification of radioligand binding, include a [3H]microscale in every cassette. Arrange the sections randomly and expose the sections for direct comparison in the same cassette.</li>
	<li>Erase the tritium-sensitive phosphor imaging plate immediately before usage in order to remove accumulated signals from storage and to eliminate background signals. Therefore, load the plate into phosphor imaging machine and expose it to visible/infrared light according to the instructions of the manufacturer.</li>
	<li>Remove the plate from 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 3 days at RT shielded from light.</li>
	<li>Because light erases signal from the imaging plate, carefully open the cassette in the dark and immediately transfer the imaging plate into the dark box of a phosphor imager or place the phosphor imager in a dark room. 
	NOTE: Make sure to notate the spatial arrangement of the slides during exposure in order to identify individual specimen on the digital image after analysis. Therefore, phosphor imaging plates also display one corner cut in a distinct angle in order to identify the correct orientation of the plate on the digital picture.</li>
	<li>Scan the plate at the highest resolution possible to obtain a digital image.</li>
</ol>
4. Optional: Cresyl Violet Staining of Tissue Sections
<ol>
	<li>Prepare 1% cresyl violet solution by mixing 5 g of cresyl violet acetate in 500 mL of deionized water (dH2O) until dissolved (approximately 2 h). Filter through a filter paper using a funnel into a new 500 mL bottle. Adjust pH to 3.5-3.8.</li>
	<li>Position the slide staining set under fume hood. Prepare trays with the following solutions in white polypropylene trays (except for xylene): 
	a. 50% ethanol/50% dH2O 
	b. 70% ethanol/30% dH2O 
	c. 100% ethanol 
	d. 100% ethanol 
	e. 100% dH2O 
	f. 1% cresyl violet 
	g. 0.07% acetic acid (add 175 &#181;L of acetic acid to 250 mL of dH2O). 
	h. 100% xylene in green solvent-resistant trays 
	i. 100% xylene in green solvent-resistant trays</li>
	<li>Transfer the slides to the fume hood and place them in a slide rack.</li>
	<li>Dissolve the lipids through increasing graded series of ethanol in dH2O into 100% ethanol (tray a-d) by dipping the slides for 1 min.</li>
	<li>Rehydrate the specimens to dH2O through descending concentrations of ethanol (tray a-d in reverse order, followed by tray e) by dipping the slides for 1 min.</li>
	<li>Immerse the specimens in cresyl violet solution for 10 min.</li>
	<li>Rinse the specimens in 0.07% acetic acid by lifting the slides up and down gently for 4-8 s. Wash the slides by dipping in dH2O for 1 min.</li>
	<li>Dehydrate the specimens by immersion of the slides for 30 s in ascending concentrations of ethanol (tray a-d).</li>
	<li>Transfer the specimens through two trays of 100% xylene (tray h and i) to quench the ethanol.</li>
	<li>Rehydrate the specimens to dH2O through descending concentrations of ethanol (tray a-d in reverse order, followed by tray e) by dipping the slides for 1 min.</li>
	<li>Remove the slides from saline with forceps. Add a few drops of organic solvent mounting media per slide and add a 24 x 60 mm coverslip on top to protect specimens. Remove air bubbles between the specimen and coverslip by gently pressing onto the coverslip. 
	NOTE: Keep the remaining slides in xylene during mounting to prevent drying.</li>
	<li>Dry the slides overnight in a fume hood at RT.</li>
	<li>Obtain a picture of specimen with a microscope and 1.25X objective.</li>
</ol>
5. Densitometric Analysis of Digital Image
<ol>
	<li>Measure relative optical densities (RODs) of each calibration standard from the [3H]microscale with an image analysis software.
	<ol>
		<li>Select an area of equal size for each point of the [3H]microscale using a tool for Region creation from the menu item Region determination. Assign a number to each selected area by clicking on Number under the menu item Label.</li>
		<li>Export OD values for each point of the calibration standard by clicking File | Export | 2D region report. Transfer ROD values to a spreadsheet and normalize by the size of the selected area. Perform linear regression to obtain a standard curve for further densitometric analysis. 
		NOTE: Make sure that the selected areas are labelled in order to identify matching ROD values and samples.</li>
	</ol>
	</li>
	<li>Perform quantification of autoradiograms using the proprietary imaging software by selecting the region of interest (ROI) using a Region creation tool in every section and measuring its ODs. Select the same region in every section by creating a template for the region of interest, which is copied and manually adjusted to minor variations in brain anatomy for each autoradiogram. Identify the anatomy of the ROI by comparison of autoradiograms with a brain atlas18. When multiple treatments are compared, perform the analysis blinded and randomized in order to avoid biased selection of ROIs.</li>
	<li>Export ROD values and sizes of selected areas into a spreadsheet by clicking File | Export | 2D region report.</li>
	<li>Divide the measured ROD of the selected ROI by its area to obtain the density per specific area.</li>
	<li>Measure the ROD of the background of the plate and export corresponding ROD values and area size into a spreadsheet. Subtract the average background signal from every ROD value of each ROI.</li>
	<li>Average the RODs of technical replicates, i.e., section replicates using tissue from the same animal.</li>
	<li>Use the standard curve to convert RODs into units of radioligand binding, i.e., nCi/mg tissue equivalents (TE). 
	NOTE: The term TE is used because standards are generated with materials simulating tissue.</li>
	<li>Express binding by conversion of nCi/mg to nmol/mg TE according to the specific activity of the radioligand (Equation 1). 
	<img alt="Equation" src="/files/ftp_upload/58879/58879eq1v2.jpg" /> (1)</li>
	<li>To obtain specific binding values, subtract non-specific binding from total binding.</li>
	<li>Average the binding of every biological replicate by using the average of the technical replicates of each animal (obtained in Step 5.6).</li>
</ol>

The authors declare no conflicts of interest.

Autoradiography is a method which provides images of radioactive decay. The technique is routinely used to study the tissue distribution of a protein of interest in vitro based on a specific pharmacological interaction between a radiolabelled compound and its target. This provides direct information about the selectivity of the ligand for the target. In vitro autoradiography may also be used for quantitative determination of pharmacological binding parameters of radioligands, such as the dissociation constant (Kd) and density of binding sites (Bmax), as well as for determining the inhibition constant (Ki) of competing ligands1,2. Compared to traditional homogenate radioligand binding, autoradiography has the advantage of being able to visualize spatial anatomy and giving succinct details of regional expression patterns3. The method of autoradiography is therefore a relevant alternative to immunocytochemistry, especially in the absence of a validated antibody. Autoradiography is easily implemented in a standard radioisotope laboratory given the availability of a suitable radioligand with the required pharmacological specificity, access to a microtome cryostat for preparing tissue sections, and a suitable imaging device that is able to analyze the distribution of radioactivity in the respective tissue sections. Notably, an important selection criterion for the radioligand is a limited amount of binding to non-target sites. This can be to other proteins, membranes or materials such as plastic or filters, and is collectively referred to as non-specific binding. Usually, non-specific binding is non-saturable but can be saturable if it involves a specific off-target protein. The best way of validating true specific binding is to compare to tissues lacking the target, e.g., genetically engineered (knock-out) tissue4.
Here, the methodology is illustrated with the autoradiographic characterization of the high-affinity binding site for &#947;-hydroxybutyric acid (GHB) in the mammalian brain. Understanding the pharmacological interaction between GHB and its binding site is of relevance as GHB is both a clinically useful drug in the treatment of narcolepsy and alcoholism5, but also a natural constituent of the mammalian brain and a recreational drug6. High-affinity GHB binding sites were first described using [3H]GHB binding to rat brain homogenate7. Over the years, further autoradiography studies with [3H]GHB and the analogue [3H]NCS-382 has showed a high density of binding sites in forebrain regions of rat8,9,10, mouse9, pig11and monkey/human brain12. However, the molecular identity and exact functional relevance of these binding sites have remained elusive.
With the intention to further characterize the binding sites, and to facilitate studies on the physiological role of GHB, multiple radioligands incorporating different isotopes endowed with different affinities have been developed ([3H]GHB, [3H]NCS-382, [3H]HOCPCA and [125I]BnOPh-GHB)13,14,15,16(reviewed in17) (Figure 1). The combination of selective high-affinity radioligands and a very high tissue density of the binding sites have allowed for the production of high-quality images using the phosphor imaging technique9,11. Along with an outline of the practical points in setting up an autoradiographic experiment and an illustration to exemplify details, the discussion section will emphasize i) the choice of radionuclide, ii) the choice of assay conditions, and iii) the use of phosphor imaging plates versus X-ray film. The overall goal of this paper is to provide technical, methodological and scientific details on the autoradiography technique for informing about tissue distribution and pharmacological analysis of protein targets.

<table><tbody><tr><td>Absolute ethanol</td><td>Merck Millipore</td><td>107017</td><td></td></tr><tr><td>Acetic acid</td><td>Sigma-Aldrich</td><td>A6283</td><td></td></tr><tr><td>BAS-TR2040 Imaging Plate</td><td>GE Healthcare Life Science</td><td>28956481</td><td>20x40 cm - Sensitive to tritium</td></tr><tr><td>Cresyl violet acetate</td><td>Sigma-Aldrich</td><td>C5042-10G</td><td></td></tr><tr><td>DPX (non-aqueous mounting medium for microscopy)</td><td>Merck Millipore</td><td>100579</td><td></td></tr><tr><td>O.C.T. Compound, 12 x 125 mL</td><td>Sakura</td><td>4583</td><td>Tissue-Tek</td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td>16005-1KG-R</td><td></td></tr><tr><td>Superfrost Plus slides</td><td>VWR</td><td>631-9483</td><td>microscope slides</td></tr><tr><td>Tissue-Tek Manual Slide Staining Set</td><td>Sakura Finetek Denmark ApS</td><td>4451</td><td></td></tr><tr><td>Tritium Standard on Glas</td><td>American Radiolabeld Chemicals, Inc.</td><td>ART 0123</td><td></td></tr><tr><td>Xylene substitute</td><td>Sigma-Aldrich</td><td>A5597</td><td></td></tr></tbody></table>

autoradiography as a simple and powerful method for visualization and characterization of pharmacological targets

In vitro autoradiography aims to visualize the anatomical distribution of a protein of interest in tissue from experimental animals as well as humans. The method is based on the specific binding of a radioligand to its biological target. Therefore, frozen tissue sections are incubated with radioligand solution, and the binding to the target is subsequently localized by the detection of radioactive decay, for example, by using photosensitive film or phosphor imaging plates. Resulting digital autoradiograms display remarkable spatial resolution, which enables quantification and localization of radioligand binding in distinct anatomical structures. Moreover, quantification allows for the pharmacological characterization of ligand affinity by means of dissociation constants (Kd), inhibition constants (Ki) as well as the density of binding sites (Bmax) in selected tissues. Thus, the method provides information about both target localization and ligand selectivity. Here, the technique is exemplified with autoradiographic characterization of the high-affinity &#947;-hydroxybutyric acid (GHB) binding sites in mammalian brain tissue, with special emphasis on methodological considerations regarding the binding assay parameters, the choice of the radioligand and the detection method.

Using the described protocol, the anatomical distribution of the high-affinity GHB binding sites was visualized with the radiolabelled GHB analogue [3H]HOCPCA in mouse brain, which was cut into coronal, sagittal and horizontal sections (Figure 3). High levels of binding were observed in hippocampus and cortex, lower binding in striatum and no binding was detected in cerebellum, corresponding to previous reported expression patterns of the high-affinity GHB sites9,10,11,12. As shown here, the anatomical structures may be visualized using different sectioning planes and anatomical integrity may be supported by cresyl violet staining. For GHB high-affinity binding sites, especially regions with low radioligand binding, such as cerebellum, are confirmed with subsequent staining of the tissue (Figure 3). Coronal sections are most often found in the literature9,10. They are practical for quantitative purposes as a higher amount of sections can be obtained from one brain. Sagittal and horizontal sections have the advantage of visualizing binding throughout most of the rodent brain within one section thus providing great overview. Figure 4illustrates the evolutionary conservation of the high-affinity GHB binding sites in the mammalian brain. [3H]HOCPCA binding sites were detected in mouse, rat as well as in pig brain tissue enabling comparison of gross brain anatomy between species. Generally, evolutionary conservation and regional distribution studies may aid significantly in the characterization of a protein of interest, in this case a novel target, and may thus give indications about its physiological function19.
The high-affinity GHB binding sites were probed with GHB radioligands, which display different affinities for the binding sites but comparable specific activities (Figure 5). [3H]HOCPCA was previously shown to have a Kd of 74 nM, which is superior to the commercially available radioligand [3H]NCS-382 with a Kd of 697 nM, both determined at pH 6.0 by quantitative autoradiography9. Thus, [3H]HOCPCA is endowed with much higher sensitivity, leading to an excellent signal-to-noise ratio. Due to the lower sensitivity of [3H]NCS-382, higher radioligand concentrations must be used in order to obtain similar levels of binding (compare y-axes in Figure 5). When compared to [3H]GHB, even higher radioligand concentrations (30 nM) are necessary to achieve comparable binding levels. This is predominantly due to the significantly lower affinity (Ki of 4.5 &#181;M) of this radioligand20. However, higher radioligand concentration also increases the level of non-specific binding9,10 with a consequently lower signal-to-noise ratio. This series of experiments highlights the importance of having a high-affinity radioligand for producing high-quality images.
Because the high-affinity GHB sites are expressed to so high levels in forebrain regions (60 pmol/mg17), the determination of pharmacological parameters by saturation curves is inherently difficult using standard tritium sensitive phosphor imaging plates due to the risk of oversaturated images. Therefore, Kd values for [3H]HOCPCA and [3H]NCS-382 have been obtained by first determining Ki values by homologous displacement curves, and then calculation of Kd (Figure 6)9. For most radioligands, an alternative would be to use isotope-dilution as is routinely done in homogenate binding assays. Moreover, Kd values have been determined at different pH values. Evidently, the high-affinity GHB sites are most efficiently labelled at pH 6.0 (Figure 6A and Figure 6D), since changing assay conditions to pH 7.4 substantially impact ligand affinity. Thus, the Kd for [3H]HOCPCA at pH 7.4 is approx. 30 times higher numerically than that at pH 6.0. Increasing the pH further results in a higher degree of non-specific binding, which becomes a caveat when using [3H]NCS-382 where only low amounts of specific binding can be obtained at pH 7.4 (Figure 6E). This in fact hinders the determination of pharmacological parameters using this radioligand at physiological pH9, again illustrating the power in having a radioligand with as high affinity as possible.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58879/58879fig1.jpg" /> 
Figure 1: Structures of radioligands targeting the high-affinity GHB binding sites. [3H]3-hydroxycyclopent-1-ene carboxylic acid ([3H]HOCPCA)14, [3H](E,RS)-6,7,8,9-tetrahydro-5-hydroxy-5H-benzocyclohept-6-ylidene acetic acid (NCS-382) ([3H]NCS-382)15 and [3H]&#947;-hydroxybutyric acid ([3H]GHB)16 as tritiated radioligands with comparable specific activity (20-40 Ci/mmol), as well as [125I]4-hydroxy-4-[4-(2-iodoben-zyloxy)phenyl]butanoate ([125I]BnOPh-GHB)13&#160;with an estimated molar activity of 2000 Ci/mmol21. The GHB structural element is highlighted in red. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58879/58879fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58879/58879fig2.jpg" /> 
Figure 2: Schematic overview of the protocol of in vitro autoradiography. (1) The animal is euthanized, tissue is dissected and snap-frozen on dry ice. Tissue is then sectioned on a cryostat, thaw-mounted onto microscope slides and (2) sections are incubated with radioligand until equilibrium binding. For determination of non-specific binding, solutions are supplemented with an unlabelled displacer of a related but not identical chemical structure. (3) Subsequently, unbound radioligand is removed by washing in assay buffer and salts are eliminated by rinsing with distilled H2O. When sections are dry, paraformaldehyde (PFA) fixation is performed to permanently establish the ligand-protein interaction. Sections are then exposed to phosphor imaging plates. (4) After sufficient exposure time, plates are scanned to obtain digital autoradiograms. (5) Ultimately, image analysis is performed using definitions of regions of interest (ROIs), and the binding is quantified. In the shown example, optical densities (ODs) are illustrated in sections of mouse cortex (left) and hippocampus (middle). Quantification is done by exposing sections together with a [3H]microscale (right). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58879/58879fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58879/58879fig3.jpg" /> 
Figure 3: Representative autoradiograms of 1 nM [3H]HOCPCA binding to mouse brain sections. (A) Radioligand binding to coronal, sagittal and horizontal tissue sections to illustrate the importance of the sectioning plane on the visibility of anatomical structures. (B) Cresyl violet staining of corresponding tissue sections to verify anatomical regions, particularly regions with low/absent radioligand binding. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58879/58879fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58879/58879fig4.jpg" /> 
Figure 4: Representative autoradiograms of 1 nM [3H]HOCPCA binding in different species. Comparison of (A) rat, (B) mouse and (C) pig in vitro autoradiograms in order to illustrate evolutionary conservation of binding sites to cortical and hippocampal regions along with gross brain anatomy. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58879/58879fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58879/58879fig5.jpg" /> 
Figure 5: Quantification of binding to the high-affinity GHB binding sites with different sensitivity to GHB radioligands by in vitro autoradiography in brain slices from mouse cortex and hippocampus. Total binding is reported as fmol/mg tissue equivalents (TE) for the radioligands (A) [3H]HOCPCA (1 nM) and (B) [3H]NCS-382 (7 nM) and (C) [3H]GHB (30 nM) (similar specific activities). Non-specific binding was not detected in the presence of 1 mM GHB or 1 mM HOCPCA for any of the radioligands (not shown). Data is presented as mean &#177; SD of four biological replicates each performed in three technical replicates. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58879/58879fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/58879/58879fig6.jpg" /> 
Figure 6: Representative results of autoradiographic determination of Kd and Bmax values by homologous competitive displacement of [3H]HOCPCA and [3H]NCS-382 in mouse cortical sections at pH 6.0 and pH 7.4 in order to illustrate the influence of pH on affinity of radioligands. (Kd and Bmax were calculated using the same compound as radioligand and competitor, through initial determination of Ki values22). (A) Autoradiograms with optimal signal-to-noise ratio for 1 nM [3H]HOCPCA binding at pH 6.0. (B) Changing buffer pH to 7.4 requires a higher [3H]HOCPCA concentration (8 nM) to reach significant binding levels. (C) Resulting log-concentration binding curves; mean &#177; SEM. (D) 5 nM [3H]NCS-382 binding at pH 6.0 results in low non-specific binding, whereas (E) 40 nM [3H]NCS-382 is insufficient to obtain binding at pH 7.4. (F) Resulting log-concentration binding curves; mean &#177; SEM. Data was obtained from 3-4 biological replicates each performed in 3-5 technical replicates, only [3H]NCS-382 experiments at pH 7.4 were performed with only 2 biological replicates. For both radioligands, 1 mM GHB was used for the determination of non-specific binding. For details on analysis and calculation of Kd and Bmax, see9. This figure has been adapted and reprinted from previous publication9&#160;with permission from Elsevier. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58879/58879fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Autoradiography as a Simple and Powerful Method for Visualization and Characterization of Pharmacological Targets at JoVE.com

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

The method of autoradiography is routinely used to study binding of radioligands to tissue sections for determination of qualitative or quantitative pharmacology.

In vitro autoradiography aims to visualize the anatomical distribution of a protein of interest in tissue from experimental animals as well as humans. The ...

autoradiography-as-simple-powerful-method-for-visualization

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

43.9K Views.  University of Copenhagen. The method of autoradiography is routinely used to study binding of radioligands to tissue sections for determination of qualitative or quantitative pharmacology.

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

In Vitro Imaging and Quantification of the Drug Targeting Efficiency of Fluorescently Labeled GnRH Analogues

GnRH analogues are effective targeting moieties and able to deliver anticancer agents selectively into malignant tumor cells which highly express GnRH receptors. However, the quantitative analysis of GnRH analogues&#39; cellular uptake and the investigated cell types in GnRH-based drug delivery systems are currently limited. Previously introduced, selectively labeled fluorescent GnRH I, -II and -III derivatives provide great detectability, and they have suitable chemical properties for reproducible and robust experiments. We also found that the appropriate up-to-date methods with these labeled GnRH analogues could offer novel information about the GnRH-based drug delivery systems. This manuscript introduces some simple and fast experiments regarding the cellular uptake of [D-Lys6(FITC)]-GnRH-I, [D-Lys6(FITC)]-GnRH-II and [Lys8(FITC)]-GnRH-III on the EBC-1 (lung), the BxPC-3 (pancreas) and on the Detroit-562- (pharynx) malignant tumor cells. In parallel with these GnRH-FITC conjugates, the cell surface level of GnRH-I receptors was also examined on these cell lines before and after the GnRH treatment by confocal laser scanning microscopy. The cellular uptake of GnRH-FITC conjugates was quantified by fluorescence-activated cell sorting. In these experiments minor differences among GnRH analogues and major differences among cell types was observed. The significant differences among cell lines are correlated with their distinct level of cell surface GnRH-I receptors. The introduced experiments contain practical methods to visualize, quantify and compare the uptake efficiency of GnRH-FITC conjugates in a time- and concentration-dependent manner on various adherent cell cultures. These results could predict the drug targeting efficiency of GnRH conjugates on the given cell culture, and offer a good basis for further experiments in the examination of GnRH-based drug delivery systems.

In Vitro Imaging and Quantification of the Drug Targeting Efficiency of Fluorescently Labeled GnRH Analogues

Selectively labeled fluorescent GnRH-I, -II and -III derivatives are reliable tools for tracking and quantifying their cellular uptake. This manuscript introduces experiments to visualize, quantify and compare the uptake efficiency of these GnRH conjugates in various cell lines.

GnRH analogues are effective targeting moieties and able to deliver anticancer agents selectively into malignant tumor cells which highly express GnRH ...

Cancer Research

High-resolution Spatiotemporal Analysis of Receptor Dynamics by Single-molecule Fluorescence Microscopy

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., kinetics, coexistence of different states and populations, transient interactions), which are typically hidden in ensemble measurements, such as those obtained with standard biochemical or microscopy methods. Thus, dynamic events, such as receptor-receptor interactions, can be followed in real time in a living cell with high spatiotemporal resolution. This protocol describes a method based on labeling with small and bright organic fluorophores and total internal reflection fluorescence (TIRF) microscopy to directly visualize single receptors on the surface of living cells. This approach allows one to precisely localize receptors, measure the size of receptor complexes, and capture dynamic events such as transient receptor-receptor interactions. The protocol provides a detailed description of how to perform a single-molecule experiment, including sample preparation, image acquisition and image analysis. As an example, the application of this method to analyze two G-protein-coupled receptors, i.e., &#946;2-adrenergic and &#947;-aminobutyric acid type B (GABAB) receptor, is reported. The protocol can be adapted to other membrane proteins and different cell models, transfection methods and labeling strategies.

This protocol describes how to use total internal reflection fluorescence microscopy to visualize and track single receptors on the surface of living cells and thereby analyze receptor lateral mobility, size of receptor complexes as well as to visualize transient receptor-receptor interactions. This protocol can be extended to other membrane proteins.

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., ...

Bioengineering

A Rhodopsin Transport Assay by High-Content Imaging Analysis

Rhodopsin misfolding mutations lead to rod photoreceptor death that is manifested as autosomal dominant retinitis pigmentosa (RP), a progressive blinding disease that lacks effective treatment. We hypothesize that the cytotoxicity of the misfolded rhodopsin mutant can be alleviated by pharmacologically stabilizing the mutant rhodopsin protein. The P23H mutation, among the other Class II rhodopsin mutations, encodes a structurally unstable rhodopsin mutant protein that is accumulated in the endoplasmic reticulum (ER), whereas the wild type rhodopsin is transported to the plasma membrane in mammalian cells. We previously performed a luminescence-based high-throughput screen (HTS) and identified a group of pharmacological chaperones that rescued the transport of the P23H rhodopsin from ER to the plasma membrane. Here, using an immunostaining method followed by a high-content imaging analysis, we quantified the mutant rhodopsin protein amount in the whole cell and on the plasma membrane. This method is informative and effective to identify true hits from false positives following HTS. Additionally, the high-content image analysis enabled us to quantify multiple parameters from a single experiment to evaluate the pharmacological properties of each compound. Using this assay, we analyzed the effect of 11 different compounds towards six RP associated rhodopsin mutants, obtaining a 2-D pharmacological profile for a quantitative and qualitative understanding about the structural stability of these rhodopsin mutants and efficacy of different compounds towards these mutants.

Here, we described a high-content imaging method to quantify the transport of rhodopsin mutants associated with retinitis pigmentosa. A multiple-wavelength scoring analysis was used to quantify rhodopsin protein on the cell surface or in the whole cell.

Rhodopsin misfolding mutations lead to rod photoreceptor death that is manifested as autosomal dominant retinitis pigmentosa (RP), a progressive blinding ...

Chemistry

Technical Aspect of the Automated Synthesis and Real-Time Kinetic Evaluation of [11C]SNAP-7941

Positron emission tomography (PET) is an essential molecular imaging technique providing insights into pathways and using specific targeted radioligands for in vivo investigations. Within this protocol, a robust and reliable remote-controlled radiosynthesis of [11C]SNAP-7941, an antagonist to the melanin-concentrating hormone receptor 1, is described. The radiosynthesis starts with cyclotron produced [11C]CO2 that is subsequently further reacted via a gas-phase transition to [11C]CH3OTf. Then, this reactive intermediate is introduced to the precursor solution and forms the respective radiotracer. Chemical as well as the radiochemical purity are determined by means of RP-HPLC, routinely implemented in the radiopharmaceutical quality control process. Additionally, the molar activity is calculated as it is a necessity for the following real-time kinetic investigations. Furthermore, [11C]SNAP-7941 is applied to MDCKII-WT and MDCKII-hMDR1 cells for evaluating the impact of P-glycoprotein (P-gp) expression on cell accumulation. For this reason, the P-gp expressing cell line (MDCKII-hMDR1) is either used without or with blocking prior to experiments by means of the P-gp substrate (&#177;)-verapamil and the results are compared to the ones observed for the wildtype cells. The overall experimental approach demonstrates the importance of a precise time-management that is essential for every preclinical and clinical study using PET tracers radiolabelled with short-lived nuclides, such as carbon-11 (half-life: 20 min).

Technical Aspect of the Automated Synthesis and Real-Time Kinetic Evaluation of [11C]SNAP-7941

Here, we represent a protocol for the fully automated radiolabelling of [11C]SNAP-7941 and the analysis of the real-time kinetics of this PET-tracer on P-gp expressing and non-expressing cells.

Positron emission tomography (PET) is an essential molecular imaging technique providing insights into pathways and using specific targeted radioligands ...

Radiosynthesis and PET/CT Imaging of 68Ga-Labelled Nanobody in Xenograft Models

Radiosynthesis, Quality Control, and Small Animal Positron Emission Tomography Imaging of 68Ga-Labelled Nano Molecules

Small animal Positron Emission Tomography (PET) and Single Photon Emission Computed Tomography (SPECT) imaging techniques are crucial in preclinical cancer research, necessitating meticulous attention to radiotracer synthesis, quality assurance, and in vivo injection protocols. This study presents a comprehensive workflow tailored to enhance the robustness and reproducibility of small animal PET experiments. The synthesis process in the radiochemistry laboratory using 68Ga is detailed, highlighting stringent quality control and assurance protocols for each radiotracer production. Parameters such as concentration, molar activity, pH, and purity are rigorously monitored, aligning with standards applicable to human studies. This methodology introduces streamlined syringe preparation and a custom-designed 30G cannula for precise intravenous injections into mice. Monitoring of animal health during scanning, including temperature and heart rate, ensures their well-being throughout the procedure. Dosages for PET and SPECT scans are predetermined to balance data acquisition with minimizing radiation exposure to animals and researchers. Similarly, CT scans employ pre-programmed settings to limit radiation exposure, especially pertinent in long-term studies assessing treatment effects. By optimizing these steps, the workflow aims to standardize procedures, reduce variability, and enhance the quality of small animal PET/SPECT/CT imaging. This resource provides valuable insights for researchers seeking to improve the accuracy and reliability of preclinical investigations in molecular imaging, ultimately advancing the field.

This paper describes the radiosynthesis, formulation, quality control of a new radiolabeled probe (i.e., 68Ga-labeled nanobody NM-02), and its use for small animal PET/CT imaging in a xenograft model.

Small animal Positron Emission Tomography (PET) and Single Photon Emission Computed Tomography (SPECT) imaging techniques are crucial in preclinical ...

Efficient Synthesis of Carbon-11 Radiotracers Using the Loop Method Methylation

Enhancing Efficiency and Radiolabeling Yields of Carbon-11 Radioligands for Clinical Research Using the Loop Method

A successful positron emission tomography imaging program involving carbon-11 radiotracers demands fast, efficient, and reliable synthesis methods, requiring an on-site cyclotron and radiochemistry group, as well as clinical staff trained to operate under the unique constraints of the carbon-11 radionuclide. This study examines the merits and advantages of a captive solvent &#39;loop method&#39; of radiolabeling four tracers with the carbon-11 radionuclide, producing the radioligands [11C]ER-176, [11C]MRB, [11C]mHED, and [11C]PiB.
The &#39;loop method&#39; is compared against the traditional reactor-based method of carbon-11 methylation in the course of synthesizing the same radiotracers on the identical automated platform. Further, a complete overview of the clinical research preparation of the [11C]ER-176 radiotracer is presented. As demonstrated by the production of [11C]ER-176, the captive solvent &#39;loop method&#39; of heterogeneous alkylation proved to be more efficient, with excellent radiochemical purity (99.6 &#177; 0.6%, n = 25), higher and more consistent radiochemical yield (end of synthesis (EOS) = 5.4 &#177; 2.2 GBq, n = 25) compared to the reactor method (EOS = 1.6 &#177; 0.5 GBq, n = 6), increased molar activity (loop method = 194 &#177; 66 GBq/&#181;mol, n = 25; reactor method = 132 &#177; 78 GBq/&#181;mol, n = 6), along with an average 5 min shorter reaction sequence.

This protocol presents a detailed procedure to prepare four carbon-11 radiotracers by way of the &#8220;loop method&#8221; [11C]methylation. We describe the procedure to synthesize [11C]ER-176, &#160;with data for three additional radiotracers: [11C]MRB, [11C]mHED, and [11C]PiB. The loop method provides an efficient synthesis with increased radiochemical yields against traditional reaction vessel [11C]methylation.

A successful positron emission tomography imaging program involving carbon-11 radiotracers demands fast, efficient, and reliable synthesis methods, ...

Receptor Autoradiography Protocol for the Localized Visualization of Angiotensin II Receptors

This protocol describes receptor binding patterns for Angiotensin II (Ang II) in the rat brain using a radioligand specific for Ang II receptors to perform receptor autoradiographic mapping.
Tissue specimens are harvested and stored at -80 &#176;C. A cryostat is used to coronally section the tissue (brain) and thaw-mount the sections onto charged slides. The slide-mounted tissue sections are incubated in 125I-SI-Ang II to radiolabel Ang II receptors. Adjacent slides are separated into two sets: &#39;non-specific binding&#39; (NSP) in the presence of a receptor saturating concentration of non-radiolabeled Ang II, or an AT1 Ang II receptor subtype (AT1R) selective Ang II receptor antagonist, and &#39;total binding&#39; with no AT1R antagonist. A saturating concentration of AT2 Ang II receptor subtype (AT2R) antagonist (PD123319, 10 &#181;M) is also present in the incubation buffer to limit 125I-SI-Ang II binding to the AT1R subtype. During a 30 min pre-incubation at ~22 &#176;C, NSP slides are exposed to 10 &#181;M PD123319 and losartan, while &#39;total binding&#39; slides are exposed to 10 &#181;M PD123319. Slides are then incubated with 125I-SI-Ang II in the presence of PD123319 for &#39;total binding&#39;, and PD123319 and losartan for NSP in assay buffer, followed by several &#39;washes&#39; in buffer, and water to remove salt and non-specifically bound radioligand. The slides are dried using blow-dryers, then exposed to autoradiography film using a specialized film and cassette. The film is developed and the images are scanned into a computer for visual and quantitative densitometry using a proprietary imaging system and a spreadsheet. An additional set of slides are thionin-stained for histological comparisons.
The advantage of using receptor autoradiography is the ability to visualize Ang II receptors in situ, within a section of a tissue specimen, and anatomically identify the region of the tissue by comparing it to an adjacent histological reference section.

Here we present a protocol to describe the localization of angiotensin II Type 1 receptors in the rat brain by quantitative, densitometric, in vitro receptor autoradiography using an iodine-125 labeled analog of angiotensin II.

This protocol describes receptor binding patterns for Angiotensin II (Ang II) in the rat brain using a radioligand specific for Ang II receptors to ...

Bioorthogonal Chemical Imaging of Cell Metabolism Regulated by Aromatic Amino Acids

Essential aromatic amino acids (AAAs) are building blocks for synthesizing new biomasses in cells and sustaining normal biological functions. For example, an abundant supply of AAAs is important for cancer cells to maintain their rapid growth and division. With this, there is a rising demand for a highly specific, noninvasive imaging approach with minimal sample preparation to directly visualize how cells harness AAAs for their metabolism in situ. Here, we develop an optical imaging platform that combines deuterium oxide (D2O) probing with stimulated Raman scattering (DO-SRS) and integrates DO-SRS with two-photon excitation fluorescence (2PEF) into a single microscope to directly visualize the metabolic activities of HeLa cells under AAA regulation. Collectively, the DO-SRS platform provides high spatial resolution and specificity of newly synthesized proteins and lipids in single HeLa cell units. In addition, the 2PEF modality can detect autofluorescence signals of nicotinamide adenine dinucleotide (NADH) and Flavin in a label-free manner. The imaging system described here is compatible with both in vitro and in vivo models, which is flexible for various experiments. The general workflow of this protocol includes cell culture, culture media preparation, cell synchronization, cell fixation, and sample imaging with DO-SRS and 2PEF modalities.

We present a protocol to directly visualize metabolic activities in cells regulated by amino acids using deuterium-oxide (heavy water D2O) probed stimulated Raman scattering (DO-SRS) microscopy, which is integrated with two-photon excitation fluorescence microscopy (2PEF).

Essential aromatic amino acids (AAAs) are building blocks for synthesizing new biomasses in cells and sustaining normal biological functions. For example, ...

Preparing a 68Ga-labeled Arginine Glycine Aspartate (RGD)-peptide for Angiogenesis

The &#945;v&#946;3 integrin is a heterodimeric adhesion molecule involved in tumor cell migration and angiogenesis. The integrin is overexpressed in angiogenic tumor endothelial cells, where it typically has a low concentration. This specific expression of &#945;v&#946;3 makes it a valid biomarker for antiangiogenic and imaging drugs. As a functional imaging modality, positron emission tomography (PET) provides information about biochemical and physiological changes in vivo, due to its unique high sensitivity at the nanomolar scale. Hence, radiometal-based PET radiopharmaceuticals have received great attention for the non-invasive quantification of tumor angiogenesis. This paper provides a systemic protocol to prepare a new radiometal-labeled peptide for the evaluation of angiogenesis. This protocol contains information about radiochemical reliability, lipophilicity, cell uptake, serum stability, and pharmacokinetic properties. The 68Ga-RGD-peptide is one of the representative PET ligands toward &#945;v&#946;3 integrin. Here, we introduce a protocol to prepare a 68Ga-RGD-peptide and the evaluation of its biological efficacy.

Preparing a 68Ga-labeled Arginine Glycine Aspartate RGD-peptide for Angiogenesis

The &#945;v&#946;3 integrin is a type of adhesion protein that is highly expressed on activated endothelial cells undergoing angiogenesis. Thus, evaluating the integrity of the integrin is of great interest in oncology. Here, we introduce a method to prepare 68Ga-labeled radiopeptides and a method to assess its biological effectiveness.

The &#945;v&#946;3 integrin is a heterodimeric adhesion molecule involved in tumor cell migration and angiogenesis. The integrin is overexpressed in ...

Medicine

Tracking Drug-induced Changes in Receptor Post-internalization Trafficking by Colocalizational Analysis

The intracellular trafficking of receptors is a collection of complex and highly controlled processes. Receptor trafficking modulates signaling and overall cell responsiveness to ligands and is, itself, influenced by intra- and extracellular conditions, including ligand-induced signaling. Optimized for use with monolayer-plated cultured cells, but extendable to free-floating tissue slices, this protocol uses immunolabelling and colocalizational analysis to track changes in intracellular receptor trafficking following both chronic/prolonged and acute interventions, including exogenous drug treatment. After drug treatment, cells are double-immunolabelled for the receptor and for markers for the intracellular compartments of interest. Sequential confocal microscopy is then used to capture two-channel photomicrographs of individual cells, which are subjected to computerized colocalizational analysis to yield quantitative colocalization scores. These scores are normalized to permit pooling of independent replicates prior to statistical analysis. Representative photomicrographs may also be processed to generate illustrative figures. Here, we describe a powerful and flexible technique for quantitatively assessing induced receptor trafficking.

Receptor trafficking modulates signaling and cell responsiveness to ligands and is, itself, responsive to cell conditions, including ligand-induced signaling. Here, we describe a powerful and flexible technique for quantitatively assessing drug-induced receptor trafficking using immunolabeling and colocalizational analysis.

The intracellular trafficking of receptors is a collection of complex and highly controlled processes. Receptor trafficking modulates signaling and ...

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

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Chapters in this video

In Vitro Imaging and Quantification of the Drug Targeting Efficiency of Fluorescently Labeled GnRH Analogues

High-resolution Spatiotemporal Analysis of Receptor Dynamics by Single-molecule Fluorescence Microscopy

A Rhodopsin Transport Assay by High-Content Imaging Analysis

Technical Aspect of the Automated Synthesis and Real-Time Kinetic Evaluation of [¹¹C]SNAP-7941

Radiosynthesis, Quality Control, and Small Animal Positron Emission Tomography Imaging of ⁶⁸Ga-Labelled Nano Molecules

Enhancing Efficiency and Radiolabeling Yields of Carbon-11 Radioligands for Clinical Research Using the Loop Method

Receptor Autoradiography Protocol for the Localized Visualization of Angiotensin II Receptors

Bioorthogonal Chemical Imaging of Cell Metabolism Regulated by Aromatic Amino Acids

Preparing a ⁶⁸Ga-labeled Arginine Glycine Aspartate (RGD)-peptide for Angiogenesis

Tracking Drug-induced Changes in Receptor Post-internalization Trafficking by Colocalizational Analysis