The work for this project was supported by grants from the National Institutes of Health R01-NR5339 and the Uniformed Services University of the Health Sciences-R061LD.

1. Isolation and Preparation of Pial Arteries
<ol>
	<li>While the rat is anesthetized insert and secure a catheter into an artery and perfuse with 1% Evans blue in 1X PBS. Pial vessels stain well with the Evans blue dye making them easier to isolate.</li>
	<li>Extract the brain and store at -70&deg;C until needed. Optimally, brains should be stored no longer than 2 months. </li>
	<li>Using a dissecting microscope remove pial arterioles (average diameter 35-50 &mu;m) from the surface of the brain . Carefully pull off all the blue-stained arterioles from dorsal and lateral the surface of the cortex using fine- tipped forceps. Remove excess adherent cortical tissue before placing in the fixative.</li>
	<li>Fix the harvested arterioles in 2% cold paraformaldehyde in 0.1M PBS for 30 min.</li>
	<li>To prepare the arterioles for sectioning pour embedding medium on the freezing disc/chuck (set at -20&deg;C). </li>
	<li>Place arteriole section flat on the chuck and wait for it to freeze. Firmly place the vessel up-right on the cryostat freezing disc on embedding media with the tip of the artery facing you. Hold it with tweezers until it is solidly frozen. </li>
	<li>Place the freezing disc with the vessel into the cryostat head and cut 8 &mu;m pial arteriole rings with the cryostat.</li>
	<li>Mount the arteriole sections on chromium-potassium-gelatin subbed slides.</li>
	<li>Store the prepared slides in a slide box in the refrigerator at 4&deg;C overnight.</li>
</ol>
2. ER-&alpha; Immunofluorescent Staining
Day 1
<ol>
	<li>Remove the slides from the refrigerator; bring each to room temperature.</li>
	<li>To wash the slides pour 1-1.5 ml 0.1M PBS (enough to cover all the sections) let sit for 10 minutes, drain and repeat procedure twice more. Each edge of the slide has a hydrophobic marker. Consequently, the liquid remains on the surface of the slide. With each washing the liquid is poured out and replaced with fresh 0.1 M PBS. </li>
	<li>Incubate slides in 1 ml of 50mM ammonium chloride for 30 min at room temperature to reduce endogenous fluorescence.</li>
	<li>Wash the slides in 0.1M PBS 3x10 min. as described in 2.2</li>
	<li>Block slides in 0.1% Triton-X 100 plus 1% normal goat serum (NGS) in PBS for 30 min. to reduce non- specific binding.</li>
	<li>Incubate samples with the primary antibody (rabbit polyclonal anti-ER-&alpha;; 1:500) in PBS + 0.1% Triton-X + 1% NGS overnight at 4&deg;C.</li>
</ol>
Day 2
<ol start="7">
	<li>Remove the slides from the refrigerator; bring each to room temperature.</li>
	<li>Wash the slides in 0.1M PBS 3x10 min. as described in 2.2</li>
	<li>Incubate with Oregon Green 488 secondary anti-rabbit in 1% NGS +0.1%Triton-X100 + PBS for 2 hour in the dark at room temperature. From this step, the next steps must be done in the dark.</li>
	<li>Wash the slides in 0.1M PBS 3x10 min. as described in 2.2</li>
	<li>Under a ventilated hood, place 1 drop of 4', 6-diamidino-2-phenylindole (DAPI) plus mounting media on the vessels and then place a cover slip over the sample.</li>
	<li>Apply clear nail polish to seal the edges of the cover slip. Do not move the slides until completely dry, which takes approximately 24 hours.</li>
</ol>
3. Digital Fluorescence Imaging
<ol>
	<li>For imaging ER-&alpha; in the pial vessel slice we use a Nikon Eclipse 80i digital fluorescent microscope with filters for 3 colors (blue, green and red) equipped with a digital camera.</li>
	<li>Insert the prepared slides with the stained pial vessel segments under the microscope and adjust to visualize the fluorescently labeled areas of interest. Start at x100 magnification then increase to x600 magnification.</li>
	<li>Use the camera to capture images in DAPI (blue) and FITC (green) channels for cell nuclei (DAPI) and ER-&alpha; (Oregon Green) using Nikon Extended Depth Focus software to convert multiple views of vessel segments into 2-D images.</li>
</ol>
4. Representative Results:
We localized estrogen receptor-&alpha; in pial arterial vessels using fluorescent probes and optimized our visualization of the receptor using digital fluorescence microscopy. To detect the presence of the receptor, a rabbit polyclonal primary antibody and an Oregon Green 488 labeled secondary antibody was used to image the receptors in pial arteries isolated from the surface of the brain. Also, a nuclear stain (DAPI) was used to identify the cell nuclei and determine if we could detect receptors located in the nucleus since ER-&alpha; has been reported to be present in the cell cyotosol and the nucleus. In addition, we conducted confirmatory experiments to validate the specificity of the primary antibody and to verify binding of the secondary to the primary antibody.
<img src="/files/ftp_upload/3203/3203fig1.jpg" alt="Figure 1"/> Figure 1. Immunofluorescent staining for ER- &alpha; (green) and counterstaining with DAPI (blue) in a pial artery ring isolated from female rat brain. Fig 1A shows the merged images suggesting the presence of some intranuclear estrogen receptor-a (arrow). Fig 1B and 1C are the focused image of ER- &alpha; which is a combination of Z-stack images. Images were taken at x600 magnification and are from the cut ring of the same vessel in different planes. Arrows indicate estrogen receptor- &alpha;.
<img src="/files/ftp_upload/3203/3203fig2.jpg" alt="Figure 2"/> Figure 2. Immunofluorescent staining for control groups in a pial artery ring isolated from female rat brain. Fig 2D shows the vessel without primary antibody. Fig 2E shows the vessel without the secondary antibody (note distinct autofluorescence along endothelial side). Images were taken at x600 magnification and are from the cut ring of the same vessel in different planes.

<ol>
	<li>Smiley, D. A., Khalil, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estrogen+compounds,+estrogen+receptors+and+vascular+cell+signaling+in+the+aging+blood+vessels.">Estrogen compounds, estrogen receptors and vascular cell signaling in the aging blood vessels.</a> Curr. Med. Chem. 16, 1863-1887 (2009).</li><li>Miller, V. M., Duckles, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+actions+of+estrogens:+functional+implications.">Vascular actions of estrogens: functional implications.</a> Pharmacol. Rev. 60, 210-241 (2008).</li><li>Duckles, S. P., Krause, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebrovascular+effects+of+oestrogen:+multiplicity+of+action.">Cerebrovascular effects of oestrogen: multiplicity of action.</a> Clin. Exp. Parmacol. Physiol. 34, 801-808 (2007).</li><li>Stirone, C., Duckles, S. P., Krause, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+forms+of+estrogen+receptor-a+incerebral+blood+vessels:+regulation+by+estrogen.">Multiple forms of estrogen receptor-a incerebral blood vessels: regulation by estrogen.</a> Am. J. Physiol. Endocrinol. Metab. 284, E184-E192 (2003).</li><li>Dan, P., Cheung, J. C., Scriven, D. R., Moore, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epitope-dependent+localization+of+estrogen+receptor-alpha,+but+not+-beta,+in+en+face+arterial+endothelium.">Epitope-dependent localization of estrogen receptor-alpha, but not -beta, in en face arterial endothelium.</a> Am. J. Physiol. Heart Circ. Physiol. 284, 1295-1306 (2003).</li><li>Fritschy, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+my+antibody+staining+specific?+How+to+deal+with+pitfalls+of+immunohistochemistry.">Is my antibody staining specific? How to deal with pitfalls of immunohistochemistry.</a> Eur. J. Neurosci. 28, 2365-2370 (2008).</li><li>Watanabe, Y., Littleton-Kearney, M. T., Traystman, R. J., Hurn, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estrogen+restores+post-ischemic+pial+and+microvascular+dilation.">Estrogen restores post-ischemic pial and microvascular dilation.</a> Am. J. Physiol. Heart and Circ. 281, H155-H160 (2001).</li><li>Li, M., Zeynalov, E., Li, X., Miyazaki, C., Koehler, R. C., Littleton-Kearney, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+estrogen+on+postischemic+pial+artery+reactivity+to+ADP.">Effects of estrogen on postischemic pial artery reactivity to ADP.</a> Microcirculation. 16, 403-413 (2009).</li><li>Qin, X. Y., Hurn, P. D., Littleton-Kearney, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estrogen+restores+postischemic+sensitivity+to+thromboxane+mimetic+U46619+in+rat+pial+artery.">Estrogen restores postischemic sensitivity to thromboxane mimetic U46619 in rat pial artery.</a> Cereb. Blood. Flow. Metab. 25, 1041-1046 (2005).</li><li>Miyazaki, C., Koelher, R. C., Littleton-Kearney, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+HET0016+and+estrogen+on+pial+artery+vasodilatory+capacity.">Effects of HET0016 and estrogen on pial artery vasodilatory capacity.</a> FASEBJ Abstracts. , (2007).</li><li>Daneshtalab, N., Dore, J. J. E., Smeda, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Troubleshooting+tissue+specificity+and+antibody+selection:+procedures+in+immunohistochemical+studies.">Troubleshooting tissue specificity and antibody selection: procedures in immunohistochemical studies.</a> J. Pharmacol. Toxical. Methods. 61, 127-135 (2010).</li><li>Lorincz, A., Nusser, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specificity+of+immunoreactants.">Specificity of immunoreactants.</a> J. Neurosci. 28, 9083-9086 (2008).</li></ol>

The views expressed are those of the authors and do not reflect the official policy or position of the Uniformed Services University of the Health Sciences, the Department of Defense, or the United States government.

Previously, we showed that after transient global ischemic brain injury the pial artery capacity to change diameter in response to both vasodilators 7, 8 and vasoconstrictors 9 is markedly depressed in young ovariectomized rats. Chronic estrogen repletion restores vasomotor responses 7-9. Further, delayed estrogen replacement reverses the beneficial effects of estrogen on pial artery vasodilatory capacity 10 leading us to question whether long-term estrogen depletion affects ER-&alpha; density in the cerebrovasculature. We planned to use immunofluorescent labeling combined with western blotting to evaluate changes in ER-&alpha; expression in response to chronic estrogen depletion. Because we had some difficulty in visualization of discrete groups of receptors we modified the method that we demonstrate here. Therefore, our primary goal in this study was to first develop a relatively simple method to optimize the visualization of the receptors in pial arterioles. In agreement with several reports 6,11,12 we found it necessary to make adjustments for the type of tissue we were probing, the sample thickness, the distribution of ER-&alpha; in the vessel, as well as the degree of non-specific binding. 
Our method is very effective to visualize the presence of ER-&alpha; in pial vessel slices. But as others have previously pointed out the successful use of these methods is highly dependent on the specificity and concentrations of the antibodies, the subcellular location of the proteins of interest, fixation and blocking protocols and tissue handling 12. Prior to visualization of our vessel preparations we spent time to work out all these variables. We have highlighted how we needed to adjust some of our approach in this paper.
We set out to optimally visualize ER-&alpha; in pial arterioles and this presented a few challenges. First, the average size of a rat pial artery is between 35-50 &mu;m making isolation and removal from the brain surface somewhat tricky. We found that perfusion with Evans blue dye prior to sacrifice makes dissection of the arteries easier. Evan's blue can affect fluorescence, but we feel that the advantage of using this dye outweigh its minor disadvantages. In our protocol the vessels are washed several times. This minimizes the residual dye in the vessels and reduces the possible effects of the dye on fluorescence. Another difficulty we encountered was that the thickness of the pial vessel segments made clear visualization of the receptors difficult. Additionally, we could not obtain particularly clear images probably due to the fact that ER-&alpha; was scattered throughout several cell layers. We tried working with longitudinal vessel slices (as others have), but due to the small pial size it was difficult to handle the vessels and prevent twisting of the vessels while mounting on slides even using a microscope. Finally, we successfully modified our technique by using a cryostat and cutting the vessel cross-wise in to 8 &mu;m thick rings. The rings are easier to handle and several can be mounted on one slide. 
Once we acquired the vessels we felt it necessary to test the effectiveness of first flash freezing the tissue then fixing the vessels as suggested by Danseshatalb and associates 11. Although we found marginally less background immunofluorescence, we also noted that the receptors stained less intensely making visualization of the receptors more difficult. Consequently, we prefer to first to freeze the entire brain and store until needed (generally between 1-2 months). We then pull off the vessels as described and fix the vessels prior to slicing with the cryostat. as we demonstrate here. 
One necessary step is to run controls with both the primary and the secondary antibody separately to determine specificity and background immunofluorescence. Estrogen receptor-&alpha; antibodies can be difficult to work with. In fact, we tried 3 different primary antibodies (1 monoclonal and 2 polyclonal) before we were satisfied with the success of our receptor staining. As part of our controls we first verified the specificity of the primary antibody by omission of the addition of the primary antibody. Figure 2D shows the vessel without primary antibody. There is no staining and just a little background signal. Since the primary antibody is polyclonal, there is some diffuse non-specific background staining. Secondly, we added the primary antibody, but not the secondary antibody (Fig. 2E). One can see a nice margin representing the autofluoresence along the endothelial borders of the vessels. This is consistent with the work of Dan and associates 5. However, neither the small points of immunofluorescence that represent ER-&alpha; , nor the granular appearance reflecting the presence of numerous receptors can be detected without the combination of both the primary and secondary antibodies. We noted a distinct pattern of autofluoresence along the endothelial margins of the vessels. These independent tests for antibody specificity are important as they confirm the specificity of the antibody for ER-&alpha; and the secondary antibody binds to the primary.
In conclusion, as demonstrated by others 4, 5 brain blood vessels contain numerous ER-&alpha; sub-type receptors. In our hands, preparing 8&mu;m cross sectional slices from isolated pial arteries enhances staining and visualization of the receptors. Traditional methods use confocal microscopy to examine the samples. Having the capacity to view serial optical sections from thick specimens is an important benefit of confocal microscopy. However, purchase of a good confocal microscope can be very expensive. By using Extended Depth (EDF) software, we found that could reduce our equipment costs. Using a fluorescent microscope with EDF we obtained clear images and are able to visualize pial arterial ER-&alpha; on several levels using Z-stacks. A significant benefit of the EDF software is that it permits image capture in a manner to effectively create a 3-D picture from the images. For laboratories that do not have access to a confocal microscope or the funds to purchase one a digital fluorescent microscope with appropriated software may be a practical and less costly option depending on the goals of the investigator.

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
 <td>EVANS BLUE</td>
 <td>Sigma</td>
 <td>E-2129</td>
 </tr>
 <tr>
 <td>PBS 10X</td>
 <td>Sigma</td>
 <td>P-5493</td>
 </tr>
 <tr>
 <td>Paraformaldehyde</td>
 <td>Fisher Scientific</td>
 <td>T353</td>
 </tr>
 <tr>
 <td>Tissue-Tek O.C.T Compound</td>
 <td>VWR</td>
 <td>25608-930</td>
 </tr>
 <tr>
 <td>Ammonium chloride</td>
 <td>Sigma</td>
 <td>A9434</td>
 </tr>
 <tr>
 <td>Triton X-100</td>
 <td>Sigma</td>
 <td>T9284</td>
 </tr>
 <tr>
 <td>Normal Goat Serum</td>
 <td>Invitrogen</td>
 <td>16210-064</td>
 </tr>
 <tr>
 <td>ER-&alpha; rabbit polyclonal antibody</td>
 <td>Santa Cruz</td>
 <td>H-184</td>
 </tr>
 <tr>
 <td>Oregon green 488 goat anti rabbit IgG</td>
 <td>Invitrogen</td>
 <td>O-11038</td>
 </tr>
 <tr>
 <td>Vectashield mounting medium for fluorescence with DAPI</td>
 <td>Vector Lab</td>
 <td>H-1200</td>
 </tr>
 </tbody>
</table>

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.

Watch this Scientific Journal Video about Imaging of Estrogen Receptor-Î± in Rat Pial Arterioles using a Digital Immunofluorescent Microscope at JoVE.com

Imaging of Estrogen Receptor-&amp;#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.

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 ...

imaging-estrogen-receptor-rat-pial-arterioles-using-digital

Research

JoVE Journal

Biology

12.4K Views.  Uniformed Services University of the Health Sciences. The goal of this article is to demonstrate a method to optimize immunofluorescent detection of estrogen receptor-α (ERα) in rat pial arterial slices using a digital immunofluorescent microscope.

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

Proper Care and Cleaning of the Microscope

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a microscope causes reduction in contrast and resolution. DIC is especially sensitive to contamination and scratches on the lens surfaces. This protocol details the procedure for keeping the microscope clean.

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a microscope causes reduction in contrast and resolution. DIC is especially sensitive to contamination and scratches on the lens surfaces. This protocol details the procedure for keeping the microscope clean.

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a ...

Major Components of the Light Microscope

The light microscope is a basic tool for the cell biologist, who should have a thorough understanding of how it works, how it should be aligned for different applications, and how it should be maintained as required to obtain maximum image-forming capacity and resolution. The components of the microscope are described in detail here.

The light microscope is a basic tool for the cell biologist, who should have a thorough understanding of how it works, how it should be aligned for different applications, and how it should be maintained as required to obtain maximum image-forming capacity and resolution. The components of the microscope are described in detail here.

The light microscope is a basic tool for the cell biologist, who should have a thorough understanding of how it works, how it should be aligned for ...

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using fluorescent-labeled reporter proteins or differential interference contrast (DIC) microscopy, allows for the examination of the temporal progression of these dynamic events which is otherwise inferred from analysis of fixed samples1,2. Moreover, the study of the developmental regulations of cellular processes necessitates conducting time-lapse experiments on an intact organism during development. The Caenorhabiditis elegans embryo is light-transparent and has a rapid, invariant developmental program with a known cell lineage3, thus providing an ideal experiment model for studying questions in cell biology4,5and development6-9. C. elegans is amendable to genetic manipulation by forward genetics (based on random mutagenesis10,11) and reverse genetics to target specific genes (based on RNAi-mediated interference and targeted mutagenesis12-15). In addition, transgenic animals can be readily created to express fluorescently tagged proteins or reporters16,17. These traits combine to make it easy to identify the genetic pathways regulating fundamental cellular and developmental processes in vivo18-21. In this protocol we present methods for live imaging of C. elegans embryos using DIC optics or GFP fluorescence on a compound epifluorescent microscope. We demonstrate the ease with which readily available microscopes, typically used for fixed sample imaging, can also be applied for time-lapse analysis using open-source software to automate the imaging process.

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

The C. elegans embryo is a powerful system for studying cell biology and development. We present a protocol for live imaging of C. elegans embryos utilizing DIC optics or fluorescence using readily available epifluorescent microscopes and open-source software.

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using ...

Setting Up a Simple Light Sheet Microscope for In Toto Imaging of C. elegans Development

Fast and low phototoxic imaging techniques are pre-requisite to study the development of organisms in toto. Light sheet based microscopy reduces photo-bleaching and phototoxic effects compared to confocal microscopy, while providing 3D images with subcellular resolution. Here we present the setup of a light sheet based microscope, which is composed of an upright microscope and a small set of opto-mechanical elements for the generation of the light sheet. The protocol describes how to build, align the microscope and characterize the light sheet. In addition, it details how to implement the method for in toto imaging of C. elegans embryos using a simple observation chamber. The method allows the capture of 3D two-colors time-lapse movies over few hours of development. This should ease the tracking of cell shape, cell divisions and tagged proteins over long periods of time.

Setting Up a Simple Light Sheet Microscope for In Toto Imaging of C. elegans Development

This protocol describes the setup of a light sheet microscope and its implementation for in vivo imaging of C. elegans embryos.

Fast and low phototoxic imaging techniques are pre-requisite to study the development of organisms in toto. Light sheet based microscopy reduces ...

Implementation of a Coherent Anti-Stokes Raman Scattering (CARS) System on a Ti:Sapphire and OPO Laser Based Standard Laser Scanning Microscope

Laser scanning microscopes combining a femtosecond Ti:sapphire laser and an optical parametric oscillator (OPO) to duplicate the laser line have become available for biologists. These systems are primarily designed for multi-channel two-photon fluorescence microscopy. However, without any modification, complementary non-linear optical microscopy such as second-harmonic generation (SHG) or third harmonic generation (THG) can also be performed with this set-up, allowing label-free imaging of structured molecules or aqueous medium-lipid interfaces. These techniques are well suited for in-vivo observation, but are limited in chemical specificity. Chemically selective imaging can be obtained from inherent vibration signals based on Raman scattering. Confocal Raman microscopy provides 3D spatial resolution, but it requires high average power and long acquisition time. To overcome these difficulties, recent advances in laser technology have permitted the development of nonlinear optical vibrational microscopy, in particular coherent anti-Stokes Raman scattering (CARS). CARS microscopy has therefore emerged as a powerful tool for biological and live cell imaging, by chemically mapping lipids (via C-H stretch vibration), water (via O-H stretch vibrations), proteins or DNA. In this work, we describe the implementation of the CARS technique on a standard OPO-coupled multiphoton laser scanning microscope. It is based on the in-time synchronization of the two laser lines by adjusting the length of one of the laser beam path. We present a step-by-step implementation of this technique on an existing multiphoton system. A basic background in experimental optics is helpful and the presented system does not require expensive supplementary equipment. We also illustrate CARS imaging obtained on myelin sheaths of sciatic nerve of rodent, and we show that this imaging can be performed simultaneously with other nonlinear optical imaging, such as standard two-photon fluorescence technique and second-harmonic generation.

Implementation of a Coherent Anti-Stokes Raman Scattering CARS System on a Ti:Sapphire and OPO Laser Based Standard Laser Scanning Microscope

Coherent anti-Stokes Raman scattering (CARS) microscopy based on inherent vibration of molecule bonds permits label-free chemically selective live cell imaging. This work presents the implementation of a complementary microscopy technique on a standard multiphoton laser scanning microscope based on a femtosecond Ti:sapphire laser and an OPO laser.

Laser scanning microscopes combining a femtosecond Ti:sapphire laser and an optical parametric oscillator (OPO) to duplicate the laser line have become ...

Visualization and Quantification of the Cell-free Layer in Arterioles of the Rat Cremaster Muscle

The cell-free layer is defined as the parietal plasma layer in the microvessel flow, which is devoid of red blood cells. The measurement of the in vivo cell-free layer width and its spatiotemporal variations can provide a comprehensive understanding of hemodynamics in microcirculation. In this study, we used an intravital microscopic system coupled with a high-speed video camera to quantify the cell-free layer widths in arterioles in vivo. The cremaster muscle of Sprague-Dawley rats was surgically exteriorized to visualize the blood flow. A custom-built imaging script was also developed to automate the image processing and analysis of the cell-free layer width. This approach enables the quantification of spatiotemporal variations more consistently than previous manual measurements. The accuracy of the measurement, however, partly depends on the use of a blue filter and the selection of an appropriate thresholding algorithm. Specifically, we evaluated the contrast and quality of images acquired with and without the use of a blue filter. In addition, we compared five different image histogram-based thresholding algorithms (Otsu, minimum, intermode, iterative selection, and fuzzy entropic thresholding) and illustrated the differences in their determination of the cell-free layer width.

This study demonstrates the surgical preparation of the rat cremaster muscle for the visualization of the in vivo cell-free layer. Considerable factors affecting the accuracy of the cell-free layer width measurement are discussed in this study.

The cell-free layer is defined as the parietal plasma layer in the microvessel flow, which is devoid of red blood cells. The measurement of the in vivo ...

A Rapid Automated Protocol for Muscle Fiber Population Analysis in Rat Muscle Cross Sections Using Myosin Heavy Chain Immunohistochemistry

Quantification of muscle fiber populations provides a deeper insight into the effects of disease, trauma, and various other influences on skeletal muscle composition. Various time-consuming methods have traditionally been used to study fiber populations in many fields of research. However, recently developed immunohistochemical methods based on myosin heavy chain protein expression provide a quick alternative to identify multiple fiber types in a single section. Here, we present a rapid, reliable and reproducible protocol for improved staining quality, allowing automatic acquisition of whole cross sections and automatic quantification of fiber populations with ImageJ. For this purpose, embedded skeletal muscles are cut in cross sections, stained using myosin heavy chains antibodies with secondary fluorescent antibodies and DAPI for cell nuclei staining. Whole cross sections are then scanned automatically using a slide scanner to obtain high-resolution composite pictures of the entire specimen. Fiber population analyses are subsequently performed to quantify slow, intermediate and fast fibers using an automated macro for ImageJ. We have previously shown that this method can identify fiber populations reliably to a degree of &#177;4%. In addition, this method reduces inter-user variability and time per analyses significantly using the open source platform ImageJ.

Here, we present a protocol for rapid muscle fiber analyses, which allows improved staining quality, and thereby automatic acquisition and quantification of fiber populations using the freely available software ImageJ.

Quantification of muscle fiber populations provides a deeper insight into the effects of disease, trauma, and various other influences on skeletal muscle ...

Detecting Estrogenic Ligands in Personal Care Products using a Yeast Estrogen Screen Optimized for the Undergraduate Teaching Laboratory

The Yeast Estrogen Screen (YES) is used to detect estrogenic ligands in environmental samples and has been broadly applied in studies of endocrine disruption. Estrogenic ligands include both natural and manmade &#34;Environmental Estrogens&#34; (EEs) found in many consumer goods including Personal Care Products (PCPs), plastics, pesticides, and foods. EEs disrupt hormone signaling in humans and other animals, potentially reducing fertility and increasing disease risk. Despite the importance of EEs and other Endocrine Disrupting Chemicals (EDCs) to public health, endocrine disruption is not typically included in undergraduate curricula. This shortcoming is partly due to a lack of relevant laboratory activities that illustrate the principles involved while also being accessible to undergraduate students. This article presents an optimized YES for quantifying ligands in personal care products that bind estrogen receptors alpha (ER&#945;) and/or beta (ER&#946;). The method incorporates one of the two colorimetric substrates (ortho-nitrophenyl-&#946;-D-galactopyranoside (ONPG) or chlorophenol red-&#946;-D-galactopyranoside (CPRG)) that are cleaved by &#946;-galactosidase, a 6-day refrigerated incubation step to facilitate use in undergraduate laboratory courses, an automated application for LacZ calculations, and R code for the associated 4-parameter logistic regression analysis. The protocol has been designed to allow undergraduate students to develop and conduct experiments in which they screen products of their choosing for estrogen mimics. In the process, they learn about endocrine disruption, cell culture, receptor binding, enzyme activity, genetic engineering, statistics, and experimental design. Simultaneously, they also practice fundamental and broadly applicable laboratory skills, such as: calculating concentrations; making solutions; demonstrating sterile technique; serially diluting standards; constructing and interpolating standard curves; identifying variables and controls; collecting, organizing, and analyzing data; constructing and interpreting graphs; and using common laboratory equipment such as micropipettors and spectrophotometers. Thus, implementing this assay encourages students to engage in inquiry-based learning while exploring emerging issues in environmental science and health.

This article presents an optimized yeast estrogen screen for quantifying ligands in Personal Care Products (PCPs) that bind estrogen receptors alpha (ER&#945;) and/or beta (ER&#946;). The method incorporates two colorimetric substrate options, a six-day refrigerated incubation for use in undergraduate courses, and statistical tools for data analysis.

The Yeast Estrogen Screen (YES) is used to detect estrogenic ligands in environmental samples and has been broadly applied in studies of endocrine ...

Isolation of Primary Rat Hepatocytes with Multiparameter Perfusion Control

Primary hepatocytes are widely used in basic research on liver diseases and for toxicity testing in vitro. The two-step collagenase perfusion procedure for primary hepatocyte isolation is technically challenging, especially in portal vein cannulation. The procedure is also prone to occasional contamination and variations in perfusion conditions due to difficulties in the assembly, optimization, or maintenance of the perfusion setup. Here, a detailed protocol for an improved two-step collagenase perfusion procedure with multiparameter perfusion control is presented. Primary rat hepatocytes were successfully and reliably isolated by taking the necessary technical precautions at critical steps of the procedure, and by reducing the operational difficulty and mitigating the variability of perfusion parameters through the adoption of a special intravenous catheter, standardized sterile disposable tubing, temperature control, and real-time monitoring and alarm system. The isolated primary rat hepatocytes consistently exhibit high cell viability (85%-95%), yield (2-5 x 108 cells per 200-300 g rat) and functionality (albumin, urea and CYP activity). The procedure was complemented by an integrated perfusion system, which is compact enough to be set up in the laminar flow hood to ensure aseptic operation.

This protocol details the use of a special intravenous catheter, standardized sterile disposable tubing, temperature control complemented by real-time monitoring, and an alarm system for two-step collagenase perfusion procedure to improve the consistency in the viability, yield, and functionality of isolated primary rat hepatocytes.

Primary hepatocytes are widely used in basic research on liver diseases and for toxicity testing in vitro. The two-step collagenase perfusion procedure ...

Application of Ultrasound and Shear Wave Elastography Imaging in a Rat Model of NAFLD/NASH

Nonalcoholic Steatohepatitis (NASH) is a condition within the spectrum of Non-Alcoholic Fatty Liver Disease (NAFLD), which is characterized by liver fat accumulation (steatosis) and inflammation leading to fibrosis. Preclinical models closely recapitulating human NASH/NAFLD are essential in drug development. While liver biopsy is currently the gold standard for measuring NAFLD/NASH progression and diagnosis in the clinic, in the preclinical space, either collection of whole liver samples at multiple timepoints during a study or biopsy of liver is needed for histological analysis to assess the disease stage.
Conducting a liver biopsy mid-study is an invasive and labor-intensive procedure, and collecting liver samples to assess disease level increases the number of research animals needed for a study. Thus, there is a need for a reliable, translatable, non-invasive imaging biomarker to detect NASH/NAFLD in these preclinical models. Non-invasive ultrasound-based B-mode images and Shear Wave Elastography (SWE) can be used to measure steatosis as well as liver fibrosis. To assess the utility of SWE in preclinical rodent models of NASH, animals were placed on a pro-NASH diet&#160;and underwent non-invasive ultrasound B-mode and shear wave elastography imaging to measure hepatorenal (HR) index and liver elasticity, measuring progression of both liver fat accumulation and tissue stiffness, respectively, at multiple time points over the course of a given NAFLD/NASH study.
The HR index and elasticity numbers were compared to histological markers of steatosis and fibrosis. The results showed strong correlation between the HR index and percentage of Oil Red O (ORO) staining, as well as between elasticity and Picro-Sirius Red (PSR) staining of livers. The strong correlation between classic ex vivo methods and in vivo imaging results provides evidence that shear wave elastography/ultrasound-based imaging can be used to assess disease phenotype and progression in a preclinical model of NAFLD/NASH.

This protocol describes the use of an enhanced ultrasound technique to non-invasively observe and quantify liver tissue changes in rodent models of nonalcoholic fatty liver disease.

Nonalcoholic Steatohepatitis (NASH) is a condition within the spectrum of Non-Alcoholic Fatty Liver Disease (NAFLD), which is characterized by liver fat ...

Methods Article

Imaging of Estrogen Receptor-α in Rat Pial Arterioles using a Digital Immunofluorescent Microscope