Name: Receptor Autoradiography Protocol for the Localized Visualization of Angiotensin II Receptors (Video) | JoVE Journal
Uploaded: 2016-06-07T14:30:00.000+00:00
Duration: 12 min 3 s
Description: Learn receptor autoradiography to localize and quantify Angiotensin II receptors in rat brain sections. This quantitative method reveals brain regions affecting behavior and cardiovascular function for therapeutic development.

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5 minutes

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 re...

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

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.

The overview of the metabolic pathway of the Renin-Angiotensin System is shown in Figure 1 and the direct focus on the Angiotensin II receptor subtypes (AT1R and AT2R) is described in Figure 2. Figure 3 displays the transfer of coronal brain sections onto microscope slides, which are then run through a receptor autoradiography procedure using a predetermined 125I-SIAng II concentration as seen in F...

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 ...

The overall goal of this procedure is to anatomically localize and quantify Angiotensin II Receptor expression in brain sections utilizing Receptor Autoradiography and the histological identification of brain structures. This method identifies the brain regions where Angiotensin II affects behavior, cognitive function, and the cardiovascular system providing insights for the development of novel therapies for the treatment of neurodegenerative and cardiovascular diseases. The main advantages of this technique are that it is quantitative, qualitative, and allows the identification of functional receptors for Angiotensin II with greater specificity than other methods.This method can also characterize the functionality of other hormone receptor systems throughout the body, such as receptors related to drugs of abuse. Immediately after harvesting, place the brain tissues in brain molds that simulate the inside of the skull and wrap the molds in aluminum foil for minus 20 degree celsius storage. After 30 minutes, transfer the tissues into a sealable freezer storage bag and store the bag at minus 80 degrees celsius.Prior to sectioning, gently remove the brain from the brain mold. To section the brain tissue, transfer the specimen of interest into a cryostat set between minus 10 and minus 18 degrees celsius. When the tissue has equilibrated to the new temperature, use a glycol and resin based medium to vertically embed a small portion of the specimen into a tissue mount to enable sectioning of the coronal plane.Load the tissue onto the microtome and firmly tighten the mount in place, then begin cutting the brain at the desired thickness, then mounting the sections onto microscope slides in a vertical orientation to apply a greater surface area of the tissue to the slide. Collect the sections in sequential sets of five. When all of the sections have been collected, allow the slides to air dry for up to one hour.Then place the samples in a plastic slide box in a self sealing freezer bag for storage at minus 20 degrees celsius. To radio label the specimens, remove the first and second slides from each set of five and mount into commercially available or 3D printed slide grips. The dash one slides are for the non-specific treatment group and the dash two slides are for the total treatment group.Invert the slides in 35 to 40 milliliters of room temperature AM5 plus their respective inhibitors in the appropriate pre-incubation Coplin jars. Running multiple sets of slides can be overwhelming. Therefore it is extremely critical to place the slides in the pre-incubation jars at four minute intervals to make sure that there is no overlap during the drying step.After 30 minutes, transfer the slides into incubation slide mailers containing 10 milliliters of AM5 supplemented with the appropriate concentration of I125 SI Angiotensin II and the respective treatment group inhibitors for 60 to 90 minutes at room temperature. Determining the exact concentration of 125I SI Ang II is critical for allowing the Angiotensin II Receptors to be compared from one day to the next. At the end of the incubation, return the slides to the slide grips, block the slides, and gently swirl them for one to two seconds in two separate containers of 400 milliliters of distilled water.After the second water wash, rinse the slides with four sequential one minute washes in 30 to 40 milliliters of AM5. After the fourth wash, gently swirl the sections for one to two seconds in four changes of ice cold distilled water. Then use four blow driers set at different angles to dry the slides with cool air for four minutes.When all of the sections are dry, transfer the slides onto a paper towel. Then use double sided tape to mount the slides, tissue side up, on a piece of cardboard for X-ray film at position and including at least one iodine 125 calibration standard slide per group. To film expose the sections, in a dark room, place the cardboard slides inside a strap back X-ray cassette and turn off the lights.Turn on the safe light and carefully open a box of X-ray film. Place one film, shiny side up with the jagged edge in the bottom right corner on top of the slides in the cassette. Then carefully close the cassette with the locking bars twisted to seal out light and store the cassette at minus 20 degrees celsius.After the appropriate exposure period, transfer the film into developer solution for two minutes followed by 30 seconds in stop bath, double distilled water with acetic acid and then five minutes in fixer solution. Wash the film in a tray of running water for 20 minutes. Transfer it into a surfectant for approximately 10 seconds and hang the film to dry.For histological analysis of the brain tissue sections, thaw the dash three sections in a slide rack. Next, wash the sections in de-ionized water for one minute followed by a 10 minute incubation in thionin stain solution and three dips in one de-ionized water container. Submerge the slides for an additional 30 second de-ionized water wash.Then wash the slides in sequential ethanol washes as indicated. After the last 100%ethanol wash, wash the slides in two containers of xylene for three and five minutes consecutively. At the end of the second xylene wash, cover the upper edge of one slide at a time with a resin base, inorganic solvent mounting medium.Mount 24 by 60 millimeter cover slips onto the slides and allow the slides to dry for 48 hours. Then scan the slides and film into the computer at 2400 DPI grayscale. To analyze the film by densitometry, after scanning, empirically outline the areas of interest on each film.This is the pseudo-color image generated after opening the scanned film in the image analysis program. Include the density, scan area, and total target area in the measurements. Adjust the scan area bars to assure that each region of interest falls between the parameters of the highlighting.Then export the data into a spreadsheet to determine the specific binding present for each sample. Calibration standard units for quantification are determined based on the date the assay was performed. After scanning the films, the calibration values are used to generate a curve based on the varying concentrations of iodine 125 standards for that specific film.The calibration and standard values are recorded in triplicate for accuracy. The distinctions between the non-specific binding and the total groups as well as the tissue labels are established by assigning each set of data to the appropriate subgroups. A higher threshold value should be set by adjusting the parameters from red to black while the lower threshold value should be set near to zero to allow a more accurate measurement of the entire scanned area of interest.For example, here the final measured areas of the paraventricular nucleus of the hypothalamus in the total versus the non-specific binding groups are compared to a thionine stained section for anatomical confirmation. Non-specific binding is the amount of radioligand bound to non Angiotensin Receptors in the presence of a saturating concentration of non-radioactive Angiotensin I Receptor ligand, whereas total binding is the amount of radioligand bound in the absence of non-radioactive Angiotensin I Receptor ligand. The non-specific binding values can then be subtracted from the total binding values to obtain the specific binding values for the Angiotensin I Receptors.Once mastered, the receptor autoradiography step can be completed in about four hours, while the film developing step takes about 30 minutes per film. While performing this procedure, it is important to keep track of time to assure that all of the slides are incubated, rinsed, and dried for precisely the same duration. Following this procedure, additional studies of brain morphology can be performed to evaluate the changes in the size of the regions displaying Angiotensin II Receptor expression.This technique has paved the way for researchers studying receptor pharmacology to determine how different brain regions are affected by various neurohormones, neurotransmitters, and drugs. After watching this video, you should have a good understanding of how to successfully perform a receptor autoradiography assay, histologically stained slides, and evaluate autoradiograms for localizing receptors to specific brain regions. Remember that working with radioactivity can be hazardous and precaution such as proper shielding, containment, exposure monitoring, and disposal should always be taken to avoid radioactive contamination.

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Neuroscience

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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.

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

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

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

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.

Automated Interactive Video Playback for Studies of Animal Communication

Learn receptor autoradiography to localize and quantify Angiotensin II receptors in rat brain sections. This quantitative method reveals brain regions affecting behavior and cardiovascular function for therapeutic development.

Receptor Autoradiography Protocol for the Localized Visualization of Angiotensin II Receptors

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