This work was supported by NIH Grant HL-113905

All animal procedures carried out for this study were approved by the Institutional Animal Care and Use Committee of Nova Southeastern University in accord with the Guide for the Care and Use of Laboratory Animals, 8th Edition (The National Academies Press, Washington, DC, 2011).
1. Preparing Tissue Sections for Receptor Autoradiography
<ol>
	<li>Upon sacrifice, harvest fresh brain tissues, and wrap in aluminum foil and place in a -20 &#176;C freezer as soon as possible. To maintain the correct shape, place brains in a brain mold that simulates the inside of the skull, wrap in aluminum foil and place in a - 20 &#176;C freezer. After 30 min, place tissues in a sealable freezer storage bag and move to a -80 &#176;C freezer for long-term storage.
	<ol>
		<li>Obtain the fresh frozen tissue specimen of interest from the -80 &#176;C freezer, and transfer to a cryostat set to a minimum of -10 &#176;C and maximum of -18 &#176;C, to avoid thawing.</li>
		<li>Place the specimen onto the tissue mount with a glycol and resin-based embedding medium, only embedding a small part of the specimen into the medium. For brain, the brain is mounted vertically to enable sectioning in the coronal plane.</li>
	</ol>
	</li>
	<li>Place the tissue mount onto the microtome within the cryostat and firmly tighten in place. Ensure the left and right side of the brain are in the same antero-postero coordinates, and that the dorso-ventral axis is perpendicular to commonly used brain atlases.</li>
	<li>Begin cutting at the desired thickness (20 &#181;m recommended) and thaw mount the sections onto microscope slides in a vertical direction to have a greater surface area of space in the slide. Collect sections onto slides in sequential sets of five, i.e. the first set of slides are labelled 1-1, 1-2, 1-3, 1-4, and 1-5 (Figure 3).</li>
	<li>After filling a set of slides with sections, allow slides to air dry for up to 1 hr, then place the slides in a plastic slide box in a self-sealing freezer storage bag, and store at -20 &#176;C.</li>
</ol>
2. Receptor Autoradiography
CAUTION: Radioactivity. Use protective attire to handle radioactivity. Disposal is depended upon establishment, and must follow guidelines to properly decay (half-life 60 days) or be picked up by a certified company.&#160;
<ol>
	<li>Remove the &#34;-1&#34; and &#34;-2&#34; slides of interest from the -20 &#176;C freezer and mount into slide grips (Figure 5). Mount the &#34;-1&#34; slides together for &#39;non-specific&#39; treatment, and the &#34;-2&#34; slides for &#39;total&#39; treatment. &#39;Non-specific&#39; jars will contain 10 &#181;M final concentrations of PD123319 an AT2R antagonist, and losartan an AT1R antagonist, in assay medium buffer (AM5) (Table 1), the &#39;total&#39; binding jars will only contain 10 &#181;M PD123319 in AM5.</li>
	<li>Invert the slide grips to place the slides into the pre-incubation Coplin jars filled with 35-40 ml of AM5, and respective inhibitors for 30 min at room temperature. Start subsequent sets in their pre-incubation bath at 4 min intervals.</li>
	<li>Immediately transfer slides from the pre incubation solution to the incubation slide mailers, containing 10 ml of AM5, plus a predetermined concentration of 125I-SI-Ang II (calculated in Figure 4) with the respective inhibitors, for 60-90 min at room temperature. If there is sufficient radioligand, 10 slides can be placed (back to back) in modified slide grips and incubated with 125I-SI-Ang II in Coplin jars.</li>
	<li>Place slides back into the slide grips (if needed), blot, and rinse by gently swirling for 1-2 sec in 400 ml of distilled water in two separate containers.</li>
	<li>Transfer the slides sequentially into four Coplin jars containing 35-40 ml of AM5 for exactly 1 min each (as illustrated in Figure 4).</li>
	<li>After the last 1 min rinse, gently swirl the sections for 1-2 sec in four changes of ice cold distilled water.</li>
	<li>Blow-dry the slides with cool air (Caution: hot air volatizes the radioactive compound) using four hair dryers set up from different angles for 4 min (Figure 5), until all the sections are dry, then place on a paper towel.</li>
	<li>Mount slides with tissues facing up onto a cardboard for apposition to X-ray film using double sided tape.</li>
	<li>Mount at least one, 125 Iodine calibration standard slide onto each cardboard (Figure 6). 
	Note: Calibration standards consist of brain paste thoroughly mixed with 125Iodine bound to a compound containing a phenol ring, compacted by centrifugation in a 1 ml tuberculin syringe, that are cryostat sectioned at the same thickness as the brain sections and thaw-mounted onto microscope slides. Alternatively, 125I calibration standards in plastic resin sectioned at 20 &#181;m thickness can be obtained commercially. The plastic resin in these standards partially shields the film from the radiation such that a tissue equivalency of ~40% should be factored into the calibration.</li>
</ol>
3. Film Exposure and Development
<ol>
	<li>Proceed to a darkroom with a strap-back X-ray cassette and autoradiography film. Open the cassette and place the cardboard with slides inside (Figure 6).
	<ol>
		<li>Turn the lights off and turn on the safelight. Carefully open the box of X-Ray film, remove one film, and place the film shiny side up (with the jagged edge on the bottom right corner) on top of the slides in the cassette. Carefully close the cassette, and twist the locking bars to seal out light (Figure 6). Expose the slides for several days to several weeks at -20 &#176;C.</li>
	</ol>
	</li>
	<li>In the darkroom, open the cassette and proceed with the film developing process.
	<ol>
		<li>Place the slides into the trays consecutively; developer for 2 min, water containing 5% glacial acetic acid for 30 sec, and fixer for 5 min. The films are the placed into a tray with running water for 20 min, then placed into Photoflo for no more than 10 sec and hung. 
		Note: The exposure time is determined empirically and can involve multiple films with different exposure times: long enough to obtain measurable signals from areas with low binding, but not so long as to saturate the film by areas with high binding. Once acceptable exposures are obtained, then proceed to the next step.</li>
	</ol>
	</li>
</ol>
4. Histology
<ol>
	<li>Prepare the thionin stain and staining reagents (Table 2, Figure 7).
	<ol>
		<li>Place the &#34;-3&#34; slides in the slide rack, and transfer in sequential order beginning with deionized water for 1 min, then thionin stain solution for 10 min followed by three dips into deionized waters, and one 30 sec wash in deionized water.</li>
		<li>Following the water, place the slide rack into ethanol washes as follows; 50%, 70%, and 90% for 30 sec, followed by two ethanol washes at 100% for 1 min each. Lastly, place the slide rack into xylene for 3 min, then transfer over to a second xylene solution for 5 min.</li>
	</ol>
	</li>
	<li>Remove one slide at a time from the last xylene bath, and cover the upper edge of the slide with a resin base in organic solvent mounting medium, and place a 24 mm x 60 mm coverslip onto the slide. Allow slides to sufficiently dry for ~48 hr and then scan into the computer at 2,400 dpi grayscale.</li>
</ol>
5. Densitometric Image Analysis
<ol>
	<li>Place the film shiny side down, with the jagged edge on the bottom left corner and scan using a proprietary scanner capable of transmitting the film density information without any distortion in the imaging system computer.</li>
	<li>Open the proprietary imaging system and utilize the calibration bars to establish calibration standards for future densitometric analysis of the images on the film (Figures 9-12).</li>
	<li>Measure areas of interest by either establishing a template or empirically outlining the areas of interest. Data is the collected and separated based on film, either control or wild type (section), and region. Make sure to include density (fmol/g), scan area, and total target area in measurements (Figure 9).
	<ol>
		<li>Adjust scan area bars by assuring that region of interest falls between parameters of highlighting (Figure 10).</li>
		<li>Export data into a spreadsheet (Table 4). Multiply the density times the total target area, then divide by the scan area in order to obtain the value for the binding of that specific area. Once this is done to all values measured, substrate the established non-specific from total to yield the specific binding present. Averages may also be performed for these values.</li>
	</ol>
	</li>
</ol>

Robert Speth has licensed antibodies to angiotensin II receptors to a commercial vendor and receives royalties from the sale of said antibodies. The other authors have nothing to disclose.

The protocol described identifies the visualization of &#39;total&#39; and &#39;non-specific&#39; binding of the radioligand in adjacent sections of coronal sections of a rodent brain previously harvested and stored at -80 &#176;C , and can be readily applicable to virtually every tissue that has anatomically resolved substructures which display differential amounts of receptors or radioligand binding sites. The procedures described within the protocol are simple and the analysis is critical for correctly interpreting results. The 20 &#181;m thickness was determined to be optimal; if the section is too thick the non-specific binding will increase, making it difficult to resolve the binding of the radioligand to its target. If the section is cut thinner, it becomes difficult to successfully cut and save sequential sections without distortion. The optimal cutting temperature will vary slightly due to the nature of the tissue and the thickness of the sections and is usually determined empirically. Once contact is made between the blade and the tissue, the orientation of the specimen can be reviewed, and realigned by moving the tissue specimen back away from the blade, and reorienting the mounting ball. When cutting a rodent brain it is critical to ensure the left and right side of the brain are in the same antero-postero coordinates, and that the dorso-ventral axis is perpendicular to commonly used brain atlases. Slide sets are pre-labelled in order to conduct studies on adjacent sections of tissues. The first sets of 5 slides are labeled 1-1, 1-2, 1-3, 1-4, and 1-5. The first cut section goes on slide 1-1 in the upper left corner (this is the upper right hand corner of the slide when it is frosted side down); the second cut section goes on slide 1-2, etc. The fifth cut section goes on slide 1-5. The sixth cut section goes on slide 1-1 to the left of the first section. After the first set of slides is filled, start another set of 5 slides, labeled 2-1, 2-2, 2-3, 2-4, and 2-5. This cycle is continued as far back into the brain as necessary. The &#34;-4&#34; and &#34;-5&#34; slides are kept as a backup or to radiolabel a different receptor.
The receptor autoradiography protocol depends upon the radioligand used and the characteristics of the receptors being surveyed to determine the concentrations of buffers, salts and inhibitors. For measurement of the AT1 subtype of Ang II receptors in this procedure, a high-affinity Iodine-125 labeled ligand, 125I-Sar1 Ile8-Ang II (125I-SI-Ang II); radiolabeled by Robert C. Speth, Ph.D. (Peptide Radioiodination Shared Resource, Georgetown University), which is also available commercially) is dissolved in AM5 buffer (Table 1); a sodium phosphate buffer with sodium chloride, EDTA and bacitracin. AM5 provides good protection of the radioligand from metallopeptidases and bacitracin-sensitive peptidases providing a physiological pH and NaCl concentration to optimize binding characteristics. Pre-incubation in AM5 exposes the tissue sections to peptidase inhibitors to minimize radioligand degradation, and also dissociates endogenous Ang II from the Ang II receptors. Losartan and PD123319, AT1R and AT2R antagonists, respectively, that saturate their target receptors are added at concentrations (10 &#181;M) to prevent the radioligand from binding to these receptors to assess non-specific binding and limit specific binding to the AT1 receptor, respectively. The desired concentration of 125I-SI-Ang II for the assay is determined and the required dilution of the stock solution is determined from a spreadsheet of the radioisotope decay rate (see protocol Table 3). Optimally a concentration of radioligand approximating the KD is used which would occupy 50% of the receptors present. However, because radioligands can be costly lower concentrations e.g. 10-20% of the KD concentration can be used. This has a potential advantage of increasing the signal to noise ratio by reducing non-specific binding more than specific binding. However, longer exposure times are required to develop an image of the binding. As noted above (section 3.4), if the exposure obtained is over or under exposed, the slides can be re-exposed to a new film for a shorter and/or longer time to achieve the desired exposure.
Densitometric image analysis can be done by a proprietary imaging system that provides an extensive compendium of the applications for autoradiography as well as other applications. Calibration standards are in fmol/g of wet tissue weight (assuming a specific gravity of one for the tissue). Each standard set, along with a background will generate a standard curve. There are several curve-fitting algorithms with and without weighting available to generate a standard curve from the relative optometric density (ROD) values for the different calibration standards. Selection of the curve that best represents the data is done empirically as the subroutine for curve fitting does not provide relative error values for the different standard curves. At RODs up to ~0.6, the standard curve is pseudolinear. However, the film begins to saturate beyond this point forming a curve that asymptotes around 0.8 to 0.9 ROD units. If the standards that are bracketing the samples are above 0.9 ROD units, it is recommended to re-expose the film for a shorter period of time to obtain more reliable values of fmol/g binding of the samples closer to the pseudolinear range of the standard curve. Values derived at this extreme of the standard curve will result in small differences in ROD for significantly larger differences in radioligand binding. Hence the ability to detect changes in binding decreases as the film approaches its saturation point.
For measurements of specific regions, recommended measures are &#39;Density&#39; in fmol/g, &#39;Scan area&#39; in pixels, and &#39;Total Target Area&#39; in pixels (Figure 9). It is important to set a threshold value as close to zero as possible (Figure 10) because variations in film density or the arbitrary aspect of the standard curve derived from the calibration standards can sometimes give values less than zero. If such values are averaged for all the pixels in the Scan Area it would derive an artificially low value. The criteria used to identify areas with binding can be very subjective, so it is best to closely pair the control and experimental brains as much as possible to reduce the incidence of subjective errors. Another challenge is to decide how many sections to average to get a final reading. A good indication of sections to analyze can be determined by following an accepted rat brain atlas, along with the stained histology slides. The size of the sampling area can also be a problem. In order to compensate, a template can be established to correct for any size discrepancies. The treatment of the sections could also affect an area of the structure that has high binding and are to be considered during analysis. This requires an alternative use of the &#39;thresholding&#39; tool in the proprietary imaging system to set a density range exceeding an arbitrary value so as to represent the anatomical characteristics of the brain region being sampled such as the paraventricular nucleus of the hypothalamus within a sampling tool that encompasses the region of interest (Figure 11, panel C). It is also possible to include the area measurement into the determination of the amount of binding. This can be done by multiplying the value for specific binding times the number of pixels that were measured. Pixels can be converted to metric units by imaging a ruler in two planes, or a rectangle of known dimensions. The number of pixels that correspond to the length or area of the rectangle can then be expressed in metric units. At 2,400 dpi resolution, ~9,000 pixels = 1 mm2 in our system.
Although the use of radioactive ligands is no longer a widely used technique; it is one of the few ways to visualize the physical distribution of receptors within the tissue specimens themselves as functional (capable of binding its innate ligand). Additionally, it can quantitate the number of receptors to enable evaluation of changes in functional receptor expression between treatment groups, between different brain regions or other structures, between different strains of animals, or animals of different ages, etc. Alternative methods are available to characterize neuroanatomical aspects of AT1 receptor expression, however, they have limited value. In situ hybridization of mRNA for AT1 receptors does not always correspond with AT1 receptor expression or distribution in the brain. While immunological techniques can approximate differences in receptor expression based upon the intensity of the staining of tissue sections, it is not possible to generate a standard curve for staining to allow for a numerical determination of receptor expression. Receptor autoradiography also provides a level of specificity for receptors (or other targets that specifically bind radioligands) that cannot be guaranteed using immunological techniques. In 2009, a series of articles were published37-43 that questioned the validity of 49 antibodies that were being used to assess 18 different G protein-coupled receptors and a transient receptor potential (TRP) channel. In essence, antibodies labeled identical bands on western blots from wild-type and knockout mice for the receptors in question. Subsequently, several groups have made similar observations using commercially available AT1 and AT2 receptor antibodies44-49. The use of a bacterial artificial chromosome which generates a reporter molecule, e.g., green fluorescent protein, driven by the AT1 promoter is an indirect means of determining the presence or absence of AT1 receptor-expressing cells in mouse (but not rat) brains based upon their having a functional AT1 receptor promoter50. It is possible to count &#34;positive cells&#34; in microscopically identified brain regions, but not to quantitate changes in the intensity of expression of the AT1 receptor in these regions. Thus at this time the only validated techniques that available to directly measure Ang II receptor expression are radioligand binding methods. And, if anatomical resolution of functional AT1 receptor protein is needed at a microscopic level, receptor autoradiography is the only technique available for such direct measurement.
Receptor autoradiography is not without limitations. The major concern is that of meeting the requirements to use radioactive materials in a research setting. It is necessary to have a radiation safety program with strict guidelines in place to assure the safety of researchers who work with radioactive materials and the safe disposition of the radioactive materials. This escalates the cost of research using radioactive materials to a point that precludes their use in many research settings. Additionally, the cost of radioligands can be substantial. Thus a receptor autoradiography experiment in which a large number of slide-mounted tissue sections are incubated in containers of radioligand can use hundreds if not thousands of dollars of radioligand. Another limitation is that tissues must be frozen prior to sectioning on a cryostat, stored for a period of time, and quickly rinsed and dried after incubation with radioligand, all of which can impair the ability of the receptors or protein of interest to bind the radioligand. For ligands that have rapid association/dissociation kinetics, it may not be possible to retain the binding of the radioligand to the receptor during the time needed to rinse away non-specifically bound radioligand. Also, with low amounts of radioligand binding long periods of film exposure - one month or more - may be required before a measureable image can form on the film. Another challenge is that addition of iodine-125 to a ligand may impair the ability of the ligand to bind to its target receptor or protein due to steric hindrance. The use of a smaller molecule, e.g., tritium (3H) eliminates the problem of steric hindrance, but tritium has such low energy and low specific activity (relative to iodine-125) that it is extremely difficult to generate a film image for most receptor populations. In light of these issues, there are many situations in which receptor autoradiography is unable to identify receptors or other molecular structures of interest.
Despite its limitations, receptor autoradiography can identify specific structures within a tissue, e.g., brain regions altered in response to experimental variables compared to a normal control brain. This knowledge can help determine the role of the AT1R for Ang II or other receptor or enzymes in the pathology of disease. This knowledge has valuable pharmacological implications, as it indicates areas to target for potential therapies.

Cardiovascular disease continues to be the leading cause of death and disability in the United States, causing more than 30% of deaths in the U.S. in 20111. The most recent statistics from the American Heart Association indicate that more than one person in three has one or more type of cardiovascular disease. Cardiovascular research continues to make strides against understanding this disease, but as generations begin getting older it is imperative to continue these efforts. The Renin-angiotensin System (RAS) plays a central role in the regulation of the cardiovascular system primarily by promoting atherosclerosis, inflammation, systemic vasoconstriction, and activation of the sympathetic nervous system (Figure 1)2-8.
The RAS is a hormonal system that is activated when juxtaglomerular cells of the kidney secrete renin into the bloodstream in response to decreased blood pressure, increased sympathetic stimulation, or decreased sodium flow by the macula densa. Renin metabolizes angiotensinogen (synthesized in the liver) to form angiotensin I (Ang I). Ang I is then metabolized by angiotensin-converting enzyme (ACE), an ectoenzyme on the luminal side of vascular endothelial cells, primarily in the lungs and kidneys, to form angiotensin II (Ang II), the main effector peptide of the RAS. Ang II is capable of activating two receptor subtypes; the type 1 receptor (AT1R) and the type 2 receptor (AT2R), both which regulate the cardiovascular system, maintain fluid and electrolyte homeostasis and are now considered to affect cognitive function and neurodegenerative disease processes8,9. A local, brain-specific RAS is reported to independently synthesize Ang II. In the brain, the precursor protein angiotensinogen is synthesized in astroglia10 converted to Ang I by a renin-like enzyme3, possibly prorenin bound to the prorenin receptor11, and subsequently converted to Ang II by angiotensin-converting enzyme which is abundantly expressed on the extracellular surface of neurons in the brain12. This intrabrain generated Ang II is the agonist for the brain AT1 and AT2 receptors that are isolated from blood-borne Ang II.
While the AT1R plays an important physiological role, it is better known for its pathophysiological effects throughout the body, primarily affecting the cardiovascular system and kidneys (Figure 2). When Ang II binds to the AT1R, it causes vasoconstriction; increasing resistance to blood flow and raising blood pressure. It also promotes the synthesis and secretion of aldosterone and vasopressin, leading to increased sodium and water retention. These effects can also induce ischemic brain damage and cognitive impairments and is linked to Parkinson&#39;s disease, Alzheimer&#39;s disease, and diabetes, as well as being recently identified to affect learning and memory13-15. There is a feedback loop in the RAS in that AT1R on the juxtaglomerular cells in the kidney inhibits renin secretion. Interestingly, the AT2R generally counter-regulates the action of AT1R, causing vasodilation, neurite outgrowth, axonal regeneration, anti-proliferation, and cerebroprotection among many others16-20. The AT2R has also been identified as a target for anti-hypertension and recently, anti-cancer drugs21. Determining the localization and density of Ang II receptors within various tissues and how they are impacted by various treatments and disease states using quantitative densitometric receptor autoradiography will help uncover the role the RAS plays in specific diseases.
Receptor autoradiography has been used for over 30 years as an effective method for indicating the presence of angiotensin II receptors and other components of the RAS in the brain and other tissues of the rat, mouse, guinea pig, dog and human under a variety of experimental conditions22-34. The importance of locating Ang II receptors within the brain is that one can apply functional neuroanatomy to the actions of Ang II in the brain, e.g., the presence of AT1R in the paraventricular nucleus of the hypothalamus (PVN) suggests a function of Ang II in the brain to stimulate vasopressin, oxytocin or corticotropin releasing hormone (CRH) release, or activation of the sympathetic nervous system. Thus, drugs that block the AT1R might decrease some of these PVN-mediated effects associated with over activity of the brain RAS. Work in progress suggests that the use of AT1R antagonists can decrease Post-traumatic Stress Disorder (PTSD)-induced release of CRH and ameliorate the symptoms of PTSD (Hurt et al., submitted for publication). The PVN, subfornical organ (SFO), and amygdala are known for regulating homeostasis, appetite/thirst, sleep, memory, emotional reactions, and are the target areas of this demonstration study. These regions were examined by collecting coronal sections of a brain on microscope slides, and treating the sections with specific inhibitors along with a specific radioligand for Ang II receptors. In this study, all materials and reagents along with suggested vendors are listed, Iodine-125 was used to radiolabel an Ang II receptor antagonist, sarcosine1, isoleucine8 Ang II (SI Ang II), which was then purified as the mono125 I-SI Ang II using HPLC methods as described previously35. The use of this high specific activity radioligand allows the visualization of areas of low, moderate and high receptor density after exposure of the radiolabeled sections to x-ray film. By calibrating the film with brain paste standards containing known amounts of Iodine-125, the specific amount of Ang II receptor binding in an area can be quantified. In experimental studies, the Ang II receptor binding in the brains of experimental subjects can be compared to that in the brains of control subjects. This can indicate whether the actions of Ang II are altered in response to a genetic condition, phenotypic abnormality, disease state or drug treatment. This knowledge can then be applied to the development of therapies to treat diseases associated with dysregulation of the RAS. Alternative techniques that identify receptor binding sites, but with reduced anatomical resolution, are binding assays that use tissue membrane preparations derived from tissue homogenates, which are incubated with the radioligand over a range of concentrations to assess radioligand binding affinity as the dissociation constant (KD) and maximal binding capacity (Bmax) of the tissue of interest.
The protocol described here can be broken down into 5 major components: Preparing Tissue Sections for Receptor Autoradiography; Receptor Autoradiography; Film Exposure and Development; Histology; and Densitometric Image Analysis.

<table><tbody><tr><td>500 ml Plastic Beakers</td><td>Fisher</td><td>02-591-30</td><td></td></tr><tr><td>24 mm x 60 mm Coverslips</td><td>Fisher</td><td>22-050-25</td><td></td></tr><tr><td>Autoradiography Imaging Film 24 mm x 30 cm</td><td>Carestream-Biomax MR Film</td><td>891-2560</td><td></td></tr><tr><td>Bacitracin (from Bacillus licheniformis)</td><td>Sigma</td><td>B-0125</td><td></td></tr><tr><td>Cardboard Sheet 8 x 11</td><td>Crescent Illustration Board #201</td><td>201</td><td></td></tr><tr><td>Coplin Jars</td><td>Fisher Scientific</td><td>E94</td><td></td></tr><tr><td>Commercial hair dryers</td><td>Conair</td><td>Model SD6X</td><td></td></tr><tr><td>Disposable Culture Tubes</td><td>Fisher</td><td>14-961-26</td><td></td></tr><tr><td>EDTA (Disodium salt, Dihydrate)</td><td>USB Corporation</td><td>15-699</td><td></td></tr><tr><td>Ethanol</td><td>Fisher</td><td>16-100-210&amp;nbsp;</td><td></td></tr><tr><td>Formulary Substitute for D-19 Developer</td><td>Photographers Formulary, Inc.&amp;nbsp;</td><td>01-0036</td><td></td></tr><tr><td>Glacial Acetic Acid</td><td>Fisher</td><td>A38 SI-212</td><td></td></tr><tr><td>Histoprep/OCT</td><td>Fisher</td><td>SH75-125D</td><td></td></tr><tr><td>Film Fixer</td><td>Kodak</td><td>5160320</td><td></td></tr><tr><td>Photo flo</td><td>Kodak</td><td>1464502</td><td></td></tr><tr><td>Losartan</td><td>Fisher/Tocris</td><td>37-985-0</td><td></td></tr><tr><td>MCID&amp;trade; Core 7.0</td><td>MCID</td><td>N/A</td><td></td></tr><tr><td>NaCl</td><td>Fisher</td><td>S271</td><td></td></tr><tr><td>Peel-A-Way slide grips</td><td>VWR</td><td>48440-003</td><td></td></tr><tr><td>Permount</td><td>Fisher</td><td>SP15-100</td><td></td></tr><tr><td>PD123319</td><td>Fisher</td><td>13-615-0</td><td></td></tr><tr><td>Premium Charged Slides, Fine Ground Edge</td><td>Premiere Microscope Slides</td><td>9308W</td><td></td></tr><tr><td>&lt;sup&gt;125&lt;/sup&gt;I Ligands</td><td>Perkin Elmer</td><td>NEX- 248</td><td></td></tr><tr><td>&lt;sup&gt;125&lt;/sup&gt;SI-Ang II&amp;nbsp;</td><td>George Washington University</td><td>Radioiodinated by Dr. Speth</td><td></td></tr><tr><td>Slide Mailers</td><td>Fisher Scientific</td><td>HS15986</td><td></td></tr><tr><td>Sodium Dibasic Phosphate Anhydrous (Na&lt;sub&gt;2&lt;/sub&gt;PO4)</td><td>Fisher</td><td>RDCS0750500</td><td></td></tr><tr><td>Sodium Acetate (Anhydrous)</td><td>Fisher</td><td>BP333-500</td><td></td></tr><tr><td>Thionin&amp;nbsp;</td><td>Fisher</td><td>T409-25</td><td></td></tr><tr><td>X-Ray Casette (10 x 12)</td><td>Spectronics Corporation</td><td>Four Square</td><td></td></tr><tr><td>Xylene</td><td>Fisher&amp;nbsp;</td><td>X3P-1GAL</td><td></td></tr></tbody></table>

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.

Watch this Scientific Journal Video about Receptor Autoradiography Protocol for the Localized Visualization of Angiotensin II Receptors at JoVE.com

Receptor Autoradiography Protocol for the Localized Visualization of Angiotensin II Receptors

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 ...

receptor-autoradiography-protocol-for-localized-visualization

Nanyang Technological University

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17.8K Views.  Nova Southeastern University. 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. 

Video: Receptor Autoradiography Protocol for the Localized Visualization of Angiotensin II Receptors

The NeuroStar TMS Device: Conducting the FDA Approved Protocol for Treatment of Depression

The Neuronetics NeuroStar Transcranial Magnetic Stimulation (TMS) System is a class II medical device that produces brief duration, pulsed magnetic fields. These rapidly alternating fields induce electrical currents within localized, targeted regions of the cortex which are associated with various physiological and functional brain changes.1,2,3
In 2007, O'Reardon et al., utilizing the NeuroStar device, published the results of an industry-sponsored, multisite, randomized, sham-stimulation controlled clinical trial in which 301 patients with major depression, who had previously failed to respond to at least one adequate antidepressant treatment trial, underwent either active or sham TMS over the left dorsolateral prefrontal cortex (DLPFC). The patients, who were medication-free at the time of the study, received TMS five times per week over 4-6 weeks.4
The results demonstrated that a sub-population of patients (those who were relatively less resistant to medication, having failed not more than two good pharmacologic trials) showed a statistically significant improvement on the Montgomery-Asberg Depression Scale (MADRS), the Hamilton Depression Rating Scale (HAMD), and various other outcome measures. In October 2008, supported by these and other similar results5,6,7, Neuronetics obtained the first and only Food and Drug Administration (FDA) approval for the clinical treatment of a specific form of medication-refractory depression using a TMS Therapy device (FDA approval K061053).
In this paper, we will explore the specified FDA approved NeuroStar depression treatment protocol (to be administered only under prescription and by a licensed medical profession in either an in- or outpatient setting).

In this article, we examine the methodology and considerations relevant to the FDA approved depression treatment protocol using the Neuronetics NeuroStar TMS device.

The Neuronetics NeuroStar Transcranial Magnetic Stimulation (TMS) System is a class II medical device that produces brief duration, pulsed magnetic ...

Automated Interactive Video Playback for Studies of Animal Communication

Video playback is a widely-used technique for the controlled manipulation and presentation of visual signals in animal communication. In particular, parameter-based computer animation offers the opportunity to independently manipulate any number of behavioral, morphological, or spectral characteristics in the context of realistic, moving images of animals on screen. A major limitation of conventional playback, however, is that the visual stimulus lacks the ability to interact with the live animal. Borrowing from video-game technology, we have created an automated, interactive system for video playback that controls animations in response to real-time signals from a video tracking system. We demonstrated this method by conducting mate-choice trials on female swordtail fish, Xiphophorus birchmanni. Females were given a simultaneous choice between a courting male conspecific and a courting male heterospecific (X. malinche) on opposite sides of an aquarium. The virtual male stimulus was programmed to track the horizontal position of the female, as courting males do in the wild. Mate-choice trials on wild-caught X. birchmanni females were used to validate the prototype's ability to effectively generate a realistic visual stimulus.

Video playback is a widely used technique in animal behavior. We created and evaluated a program that applies rules-based, interactive playback of 3-D computer animations in response to real-time, automated data on subject behavior.

Video playback is a widely-used technique for the controlled manipulation and presentation of visual signals in animal communication. In particular, ...

Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate a method to fabricate suction electrodes that are used to examine CPG activity, or fictive locomotion, in dissected rodent spinal cords. The rodent spinal cords are placed in artificial cerebrospinal fluid and the ventral roots are drawn into the suction electrode. The electrode is constructed by modifying a commercially available suction electrode. A heavier silver wire is used instead of the standard wire given by the commercially available electrode. The glass tip on the commercial electrode is replaced with a plastic tip for increased durability. We prepare hand drawn electrodes and electrodes made from specific sizes of tubing, allowing consistency and reproducibility. Data is collected using an amplifier and neurogram acquisition software. Recordings are performed on an air table within a Faraday cage to prevent mechanical and electrical interference, respectively.

A demonstration of the fabrication and use of an extracellular suction electrode used to measure electrophysiological recordings of neonatal rodent spinal cords in vitro

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate ...

Nitropeptide Profiling and Identification Illustrated by Angiotensin II

Protein nitration is one of the most important post-translational modifications (PTM) on tyrosine residues and it can be induced by chemical actions of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in eukaryotic cells. Precise identification of nitration sites on proteins is crucial for understanding the physiological and pathological processes related to protein nitration, such as inflammation, aging, and cancer. Since the nitrated proteins are of low abundance in cells even under induced conditions, no universal and efficient methods have been developed for the profiling and identification of protein nitration sites. Here we describe a protocol for nitropeptide enrichment by using a chemical reduction reaction and biotin labeling, followed by high resolution mass spectrometry. In our method, nitropeptide derivatives can be identified with high accuracy. Our method exhibits two advantages compared to the previously reported methods. First, dimethyl labeling is used to block the primary amine on nitropeptides, which can be used to generate quantitative results. Second, a disulfide bond containing NHS-biotin reagent is used for the enrichment, which can be further reduced and alkylated to enhance the detection signal on a mass spectrometer. This protocol has been successfully applied to the model peptide Angiotensin II in the current paper.

Proteomic profiling of tyrosine-nitrated proteins has been a challenging technique due to the low abundance of the 3-nitrotyrosine modification. Here we describe a novel approach for nitropeptide enrichment and profiling by using Angiotensin II as the model. This method can be extended for other in vitro or in vivo systems.

Protein nitration is one of the most important post-translational modifications (PTM) on tyrosine residues and it can be induced by chemical actions of ...

Biochemistry

Visualizing Leukocyte Rolling and Adhesion in Angiotensin II-Infused Mice: Techniques and Pitfalls

Epifluorescence intravital video microscopy (IVM) of blood vessels is an established method to evaluate the activation of immune cells and their ability to role and adhere to the endothelial layer. Visualization of circulating cells by injection of fluorescent dyes or fluorophore-coupled antibodies is commonly used. Alternatively, fluorescent reporter mice can be used. Interactions of leukocytes, in particular lysozyme M+ (LysM+) monocytes, with the vessel wall play pivotal roles in promoting vascular dysfunction and arterial hypertension. We here present the technique to visualize and quantify leukocyte rolling and adhesion in carotid arteries in angiotensin II (AngII)-induced hypertension in mice by IVM.
The implantation of a catheter damages the vascular wall and leads to altered blood cell responses. We compared different injection techniques and administration routes to visualize leukocytes in a LysMCre+IRG+ mouse with widespread expression of red fluorescent protein and conditional expression of green fluorescent protein in LysM+ cells. To study LysM+ cell activation, we used AngII infused mice in which rolling and adhesion of leukocytes to the endothelium is increased. We either injected acridine orange using a jugular catheter or directly though the tail vein and compared the amount of rolling and adhering cells. We found that jugular catheter implantation per se increased the number of rolling and adhering LysM+ cells in sham-infused LysMCre+IRG+ mice compared to controls. This activation was augmented in AngII-infused mice. Interestingly, injecting acridine orange directly through the tail vein did not increase LysM+ cell adhesion or rolling in sham-infused mice. We thereby demonstrated the importance of transgenic reporter mice expressing fluorescent proteins to not interfere with in vivo processes during experimentation. Furthermore, tail vein injection of fluorescent tracers might be a possible alternative to jugular catheter injections.

This manuscript describes the use of transgenic reporter mice and different administration routes of fluorescent dyes in angiotensin II-induced hypertension using intravital video microscopy of blood vessels to evaluate the activation of immune cells and their ability to roll and adhere to the endothelium.

Epifluorescence intravital video microscopy (IVM) of blood vessels is an established method to evaluate the activation of immune cells and their ability ...

Immunology and Infection

Imaging of Estrogen Receptor-&alpha; in Rat Pial Arterioles using a Digital Immunofluorescent Microscope

Many of estrogen's effects on vascular reactivity are mediated through interaction with estrogen receptors 1, 2, 3. Although two sub-types exist (estrogen receptor -&alpha; and &beta;),estrogen receptor-&alpha; has been identified in both the smooth muscle and in endothelial cells of pial arterial segments using fluorescent staining combined with confocal laser scanning microscopy 4. Furthermore, ER-&alpha; is located in the nuclei and in the cytoplasm of rat basilar arteries 5. The receptors are abundant and fluoresce brightly, but clear visualization of discrete groups of receptors is difficult likely due to the numbers located in many cell layers of pial vessel segments. Additionally, many reports using immunohistochemical techniques paired with confocal microscopy poorly detail the requirements critical for reproduction of experiments 6. Our purpose for this article is to describe a simple technique to optimize the staining and visualization of ER-&alpha; using cross-sectional slices of pial arterioles obtain from female rat brains. We first perfuse rats with Evans blue dye to easily identify surface pial arteries which we isolate under a dissecting microscope. Use of a cryostat to slice 8 &mu;m cross sections of the arteries allows us to obtain thin vessel sections so that different vessel planes are more clearly visualized. Cutting across the vessel rather than use of a small vessel segment has the advantage of easier viewing of the endothelial and smooth muscle layers. In addition, use of a digital immunofluorescent microscope with extended depth software produces clear images of ten to twelve different vessel planes and is less costly than use of a confocal laser scanning microscope.

Imaging of Estrogen Receptor-&#945; in Rat Pial Arterioles using a Digital Immunofluorescent Microscope

The goal of this article is to demonstrate a method to optimize immunofluorescent detection of estrogen receptor-&#945; (ER&#945;) in rat pial arterial slices using a digital immunofluorescent microscope.

Many of estrogen's effects on vascular reactivity are mediated through interaction with estrogen receptors 1, 2, 3. Although two sub-types exist (estrogen ...

Biology

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.

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 ...

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

Live Cell Imaging and 3D Analysis of Angiotensin Receptor Type 1a Trafficking in Transfected Human Embryonic Kidney Cells Using Confocal Microscopy

Live-cell imaging is used to simultaneously capture time-lapse images of angiotensin type 1a receptors (AT1aR) and intracellular compartments in transfected human embryonic kidney-293 (HEK) cells following stimulation with angiotensin II (Ang II). HEK cells are transiently transfected with plasmid DNA containing AT1aR tagged with enhanced green fluorescent protein (EGFP). Lysosomes are identified with a red fluorescent dye. Live-cell images are captured on a laser scanning confocal microscope after Ang II stimulation and analyzed by software in three dimensions (3D, voxels) over time. Live-cell imaging enables investigations into receptor trafficking and avoids confounds associated with fixation, and in particular, the loss or artefactual displacement of EGFP-tagged membrane receptors. Thus, as individual cells are tracked through time, the subcellular localization of receptors can be imaged and measured. Images must be acquired sufficiently rapidly to capture rapid vesicle movement. Yet, at faster imaging speeds, the number of photons collected is reduced. Compromises must also be made in the selection of imaging parameters like voxel size in order to gain imaging speed. Significant applications of live-cell imaging are to study protein trafficking, migration, proliferation, cell cycle, apoptosis, autophagy and protein-protein interaction and dynamics, to name but a few.

Here we present a protocol to image cells expressing green fluorescent protein-tagged angiotensin type 1a receptors during endocytosis initiated by angiotensin II treatment. This technique includes labeling lysosomes with a second fluorescent marker, and then utilizing software to analyze the co-localization of receptor and lysosome in three dimensions over time.

Live-cell imaging is used to simultaneously capture time-lapse images of angiotensin type 1a receptors (AT1aR) and intracellular compartments in ...

Direct Detection of Isolevuglandins in Tissues Using a D11 scFv-Alkaline Phosphatase Fusion Protein and Immunofluorescence

Isolevuglandins (IsoLGs) are highly reactive gamma ketoaldehydes formed from H2-isoprostanes through lipid peroxidation and crosslink proteins leading to inflammation and various diseases including hypertension. Detection of IsoLG accumulation in tissues is crucial in shedding light on their involvement in the disease processes. However, measurement of IsoLGs in tissues is extremely difficult, and currently available tools, including mass spectrometry analysis, are laborious and extremely expensive. Here we describe a novel method for in situ detection of IsoLGs in tissues using alkaline phosphatase-conjugated D11 ScFv and a recombinant phage-display antibody produced in E. coli by immunofluorescent microscopy. Four controls were used for validating the staining: (1) staining with and without D11, (2) staining with bacterial periplasmic extract with the alkaline phosphatase linker, (3) irrelevant scFV antibody staining, and (4) competitive control with IsoLG prior to the staining. We demonstrate the effectiveness of the alkaline phosphatase-conjugated D11 in both human and mouse tissues with or without hypertension. This method will likely serve as an important tool to study the role of IsoLGs in a wide variety of disease processes.

This article provides a detailed methodology for the measurement of isolevuglandins in tissues by immunofluorescence using alkaline phosphatase-conjugated ScFv D11 antibody. Hypertension models in both mice and humans are used to explain the step-by-step procedures and fundamental principles associated with isolevuglandin measurement in tissue samples.

Isolevuglandins (IsoLGs) are highly reactive gamma ketoaldehydes formed from H2-isoprostanes through lipid peroxidation and crosslink proteins leading to ...

Receptor Autoradiography Protocol for the Localized Visualization of Angiotensin II Receptors

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