This study was supported by the National Institute of Health (NIH) grant R01DK109001 (to K.S.).

All procedures containing animal subjects have been approved by the Animal Welfare Committee of University of Texas Health Science Center at Houston (animal protocol number: AWC-18-0057).
1. Reagent Preparation
<ol>
	<li>1x phosphate-buffered saline (PBS, pH 7.4): to make 1 L of 1x PBS, dissolve 8 g of NaCl, 0.2 g of KCl, 1.44 g of Na2HPO4, and 0.24 g of KH2PO4 in 800 mL of distilled water. Adjust the pH to 7.4 and fill with distilled water to achieve a final volume of 1 L.</li>
	<li>1% paraformaldehyde in 1x PBS (1% PFA, wt/vol): to achieve a final volume of 50 mL, add 3.125 mL of 16% PFA (see Table of Materials) to 46.875 mL of 1x PBS. Mix well and store at 4 &#176;C for later use. 
	CAUTION: Paraformaldehyde is toxic. All procedures should be carried out under a fume hood to avoid inhalation and skin contact.</li>
	<li>0.1% polysorbate 20 (see Table of Materials) in 1x PBS (1x PBST, pH 7.4, vol/vol): to achieve a final volume of 50 mL, add 0.05 mL of polysorbate 20 to 49.95 mL of 1x PBS. Vortex and store at 4 &#176;C for up to 1 month.</li>
	<li>1% octoxynol-9 (see Table of Materials) in 1x PBS (1x PBS-TX, pH 7.4, vol/vol): to achieve a final volume of 10 mL, add 0.1 mL of octoxynol-9 to 9.9 mL of 1x PBS. Vortex and store at 4 &#176;C for up to 1 month.</li>
	<li>2% sodium azide (wt/vol): to produce 10 mL of 2% sodium azide, add 0.2 g of sodium azide to 10 mL of 1x PBS. Mix well and store at 4 &#176;C for later use.</li>
	<li>90% glycerol in 1x PBS (vol/vol): to achieve a final volume of 10 mL, add 1 mL of 1x PBS to 9 mL of glycerol. Vortex and store at room temperature (RT) for later use.</li>
</ol>
2. Animal Ddissection and Adipose Tissue Collection
<ol>
	<li>Use 6-week-old C57BL/6J male mice. Anesthetize the mice with isoflurane (4%&#8211;5% for induction then 1%&#8211;2% for maintenance). Once anesthetized, perform a toe-pinch reflex to determine the anesthetic depth, judging by the lack of pedal reflexes. Once the mice are deeply anesthetized, dissect the mice following the protocol as previously published15.</li>
	<li>Sterilize the blunt scissors and surgical area of the chest (no need to shave the hair). Open the chest by cutting the diaphragm and ribs along the lateral surface with a size of ~2 cm using blunt scissors. Clamp the chest flag with hemostat and reflect it by putting the hemostat over the head.</li>
	<li>Make a small incision into the left ventricle (~0.5 cm) with iris scissors. Insert an olive-tipped perfusion needle through the incision site and clamp the needle tip in place with the hemostat. Attach the needle to a 50 mL syringe containing paraformaldehyde fixation solution (1% PFA, pH 7.4).</li>
	<li>Open the right atrium by section cutting with iris scissors as the perfusion outlet. Start the perfusion by gently and continuously pushing the syringe at a perfusion rate close to 10 mL/min. Stop pushing when the fluid exiting the outlet is clear of any blood. The whole process should require ~5 min. 
	CAUTION: Perform these steps under a fume hood to prevent toxicity from PFA.</li>
	<li>Dissect white and brown adipose tissues from the mice. Use scissors to break the tissue samples into small pieces of chunks approximately 5 x 5&#160;x 3 mm3. Transfer the small chunks with the sterilized tweezers into the tissue embedding cassettes with proper labeling of the tissue samples on the cassettes.</li>
	<li>Immerse the embedding cassettes containing tissue samples into the fixation solution (1% PFA in 1x PBS, pH 7.4) at 4 &#176;C for 24&#8211;48 h. 
	NOTE: The fixation solution and fixation time vary based on sizes of the tissues16,17,18.</li>
	<li>Wash the samples with fresh 1x PBS buffer three times (0.5 mL/wash). 
	CAUTION: Perform this step under a fume hood. The paraformaldehyde waste should be properly handled according to hazardous waste disposal procedures. 
	NOTE: The tissues can be stored in 1x PBS at 4 &#176;C for further processing.</li>
</ol>
3. Antibody Incubation
<ol>
	<li>Cut the fixed tissue samples into approximately 2 mm3 cubes with the sterilized scissors. For permeabilization, transfer the samples into a 1.5 mL tube containing 1 mL of 1x PBS-TX (1% octoxynol-9 in 1x PBS, see Table for Materials, pH 7.4) and gently rotate the tubes at RT for 1 h at 18 rpm.</li>
	<li>Carefully remove the 1x PBS-TX by aspiration. Wash the samples 3x by adding the 1x PBS directly into the same tubes (no need to change the tubes). During each wash, invert the tubes several times.</li>
	<li>For blocking, add 0.5 mL of blocking buffer (Table of Materials) to the samples and incubate at RT for 2 h with gentle rotation.</li>
	<li>To prepare 0.4&#160;mL of primary antibody solution, dilute 2&#160;&#956;L of anti - endomucin (the marker of blood vessels) 5 (1:200) and 2&#160;&#956;L of anti &#8212; tyrosine hydroxylase (TH, the marker of nerve fibers) 5 (1:200, 1 &#181;g/mL) antibodies (Table of Materials for antibody information) into 396&#160;&#956;L of blocking buffer. Vortex and spin down to recover the volume.</li>
	<li>For the first antibody incubation, carefully remove the blocking buffer from the tissue chunks. Add 100 &#956;L of primary antibody solution prepared in step 3.4 into tubes and incubate at 4 &#176;C overnight. 
	NOTE: The antibody dilution and incubation time may vary among different antibodies. It is recommended to add 0.02% sodium azide to the antibody solution to prevent microbial growth. To prepare 0.4&#160;mL of primary antibody solution with sodium azide, add 4&#160;&#956;L of antibodies (1 &#956;L of each) and 4&#160;&#956;L of 2% sodium azide into 392&#160;&#956;L of blocking buffer.</li>
	<li>Carefully collect the primary antibody solution for reuse if desired. Wash the samples with 1x PBST (pH 7.4, 100 &#181;L/wash) three times (30 min each) with gentle rotation at 18 rpm.</li>
	<li>For the preparation of secondary antibody solution, dilute 2&#160;&#956;L of fluorophore (wave length 495 nm) conjugated anti-goat IgG (1:200) and 2&#160;&#956;L of fluorophore (wave length 650 nm) conjugated anti-rabbit IgG (1:200) (see Table of Materials) into 396&#160;&#956;L of blocking buffer. Vortex and spin briefly to collect all the liquid. 
	NOTE: Protect the samples from light during the following procedures.</li>
	<li>For the second antibody incubation, remove the last wash buffer from samples. Add 100 &#956;L of secondary antibody solution into tubes and incubate at RT for 2 h with gentle rotation at 18 rpm.</li>
	<li>Carefully remove the secondary antibody solution. Wash the samples 3x with 1x PBST (pH 7.4, 30 min each) with gentle rotation at 18 rpm. 
	NOTE: Do not reuse this secondary antibody solution, as it may be contaminated with the trace amounts of primary antibodies brought by the tissues.</li>
	<li>For optical clearance, immerse the samples in 1 mL of 90% glycerol and keep the samples at 4 &#176;C in darkness until they become transparent. 
	NOTE: The immersion duration depends on the tissue type and size. Typically, a 2 mm3 cube of white adipose tissue needs overnight incubation, while the same size brown adipose tissue sample needs longer.</li>
	<li>Adhere a silicone isolator to the slide to create a well for volume imaging. Alternatively, attach several layers of transparent tape to the slides and cut a square out of the middle to create a well (Figure 2). 
	NOTE: Adjust the well depth to fit the sample size. In this protocol, a 1 mm well depth is used.</li>
	<li>Carefully transfer the samples into the well and fill it with the mounting medium (see Table of Materials). Lay a cover slip on the surface and seal the corners of the cover slip with high viscosity medium (see Table of Materials). Let the mounting medium cure for 24 h at RT in darkness. 
	NOTE: Ensure that no space is left between the sample and cover slip. Avoid bubbles under the cover slip. Avoid placing excessive pressure on the samples.</li>
</ol>
4. Image Acquisition
<ol>
	<li>Acquire Z-stack images (refer to steps 4.2&#8211;4.7) with the 20x objective of a confocal microscope/software.</li>
	<li>Start the system. Click on the &#60;Configuration&#62; tab and activate the argon laser and HeNe 633 laser. Click on the &#60;Acquire&#62; tab. In the &#60;Visible&#62; laser lines panel, move the corresponding intensity sliders up to choose the laser lines of 488 nm and 633 nm. Initially, the intensities can be set to 20%&#8211;30%. Select the 488/561/633 triple dichoric mirror. Activate the PMTs by clicking on the &#60;Active&#62; checkboxes, choosing PMT1 for the emission excited by the 488 nm laser and PMT3 for that of the 633 nm laser. Set the wavelength range to 500&#8211;550 nm for PMT1 and 650-750nm for PMT3. Select the psudocolor to be used for image display by double clicking the colored rectangle beside each PMT and choosing a color from the pop-up menu.</li>
	<li>Click on &#60;Live&#62; to check the image of samples. Use the z-Position knob to select a plane of focus within the XY region of interest. Adjust the brightness of the images by tuning the laser intensity, smart gain and offset. For each fluorescence channel, perform this adjustment under the &#60;QLUT&#62; (Quick Look Up Table) display mode. Click on the &#60;QLUT&#62; button to enter the QLUT mode, in which saturated pixels are displayed in blue to aid the setting of appropriate brightness level. Click twice on the &#60;QLUT&#62; button to go back to the pseudocolor display mode. 
	NOTE: The numerical values of the setting may vary among each experiment.</li>
	<li>While scanning, turn the Z-Position knob counterclockwise to move the plan of focus to one end of the volume of interest and then click on the &#60;End&#62; arrowhead to set the scanning end position. Then turn the Z-Position knob clockwise to move the plane of focus through the specimen to the other end of the volume of interest and then click on the &#60;Begin&#62; arrowhead to set the scanning begin position. 
	NOTE: For the comparison between different samples, define the same Z-volume for different samples.</li>
	<li>Adjust the z-step size to 3 &#181;m. Change the image quality by selecting a format of 1024 x 1024, speed of 100 Hz, and line average of 2.</li>
	<li>Select &#60;Start&#62; to initiate the z-stack image acquisition.</li>
	<li>For maximum projection of the acquired image stack, click on &#60;Process&#62; tab and then &#60;Tool&#62;, select &#60;3D Projection&#62; and enter &#60;Maximum&#62; in the method list without changes to X, Y and Z. Set &#60;Threshold&#62; to 0. Click on &#60;Apply&#62;. The maximum intensity of the Z-volume will be stacked into a 2D image which will be shown&#160;(Figure 4A).</li>
</ol>
5. Analysis of Blood Vessel and Nerve Fiber Networks
<ol>
	<li>Analysis of 2D images via open source software (see Table of Materials)19
	<ol>
		<li>Open the stacked images in the software. Adjust Vessel diameter and intensity until all the vessel structures can be properly selected (Figure&#160;4B).</li>
		<li>Select Run analysis and export the data following guidance of the software.</li>
	</ol>
	</li>
	<li>Analysis of 3D images via the licensed software (see Table of Materials)
	<ol>
		<li>Open the raw data of images with the software. Adjust the threshold and other parameters until the vessel structures in each layer of the z-volume are properly selected (refer to the user guide for details in the software) (Figure 4C).</li>
		<li>Analyze the selected segments characters and export the data following the guidance of the software.</li>
	</ol>
	</li>
</ol>

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The authors declare that they have no competing financial interests.

Adipose tissue remodeling is directly linked to metabolic dysregulation during obesity development1,2. Angiogenesis and sympathetic innervation are both essential for the dynamic remodeling process2,12. Therefore, developing an applicable approach to visualize the new blood vessels as well as nerve fibers are of great importance. Previous methods have been reported for documenting angiogenesis in adipose ...

Adipose tissue has key metabolic and endocrine functions1. It dynamically expands or shrinks in response to different nutrient stresses2. The active tissue remodeling process consists of multiple physiological paths/steps including angiogenesis, fibrosis, and shaping of local inflammatory microenvironments2,3,4. Some physical stimuli, such as cold exposure and exercise, may trigger sympathetic activation, which ultimately leads to new blood vessel formation and sympathetic innervation in adipose tissues5,6. These remodeling processes are linked tightly to systemic metabolic outcomes including insulin sensitivity, the hallmark of type 2 diabetes2. Thus, visualization of these pathological changes is very important to understanding the healthy status of whole adipose tissues.
Angiogenesis is the process of new blood vessel formation. Since blood vessels provide oxygen, nutrients, hormones, and growth factors to tissue, angiogenesis has been considered a key step in adipose tissue remodeling, which has been documented with different techniques6,7,8,9,10,11,12,13. However, there remain questions about the resolution of the images, efficiency of immunostaining, and methods for quantification of vessel density. Compared to new blood vessel formation, innervation in adipose tissue has been underestimated for a long time. Recently, Zeng et al.14 used advanced intravital two-photon microscopy and demonstrated that adipocytes are surrounded by layers of nerve fibers14. Since then, researchers have started to appreciate the pivotal role of sympathetic innervation in regulation of adipose tissue physiology. Thus, developing an easy and practical approach to document adipose nerve innervation is important.
Here, we report an optimized method for the co-staining of blood vessels and nerve fibers based on our previous protocols. With this method, we can achieve clear images of blood vessels and nerve fibers without noisy background. Moreover, we obtain a resolution that is high enough for performing quantitative measurement of densities with open source software. By using this new approach, we can successfully compare the structures and densities of blood vessels and nerve fibers in different adipose depots.

<table><tbody><tr><td>Alexa Fluor 488 AffiniPure Bovine Anti-Goat IgG (H+L)</td><td>Jackson ImmunoResearch</td><td>805-545-180</td><td>Lot: 116969</td></tr><tr><td>Alexa Fluor 647 AffiniPure Donkey Anti-Rabbit IgG (H+L)</td><td>Jackson ImmunoResearch</td><td>711-605-152</td><td>Lot: 121944</td></tr><tr><td>Amira 6.0</td><td>Thermo Fisher Scientific</td><td></td><td>Licensed software</td></tr><tr><td>Angio tool</td><td>National Institutes of Health</td><td></td><td>Open source software&lt;br/&gt; https://ccrod.cancer.gov/confluence/display/ROB2/Home</td></tr><tr><td>Anti-mouse endomucin antibody</td><td>R&amp;amp;D research system</td><td>AF4666</td><td>Lot: CAAS0115101</td></tr><tr><td>Anti-tyrosine hydroxylase antibody</td><td>Pel Freez Biologicals</td><td>P40101-150</td><td>Lot: aj01215y</td></tr><tr><td>Cover glasses high performance, D = 0.17 mm</td><td>Zeiss</td><td>474030-9020-000</td><td></td></tr><tr><td>Cytoseal 280</td><td>Thermo Fisher Scientific</td><td>8311-4</td><td>High-viscosity medium</td></tr><tr><td>Glycerol</td><td>Fisher</td><td>G33-500</td><td></td></tr><tr><td>Paraformaldehyde,16%</td><td>TED PELLA</td><td>170215</td><td></td></tr><tr><td>Press-to-Seal Silicone Isolator with Adhesive, eight wells, 9 mm diameter, 1.0 mm deep</td><td>INVITROGEN</td><td>P24744</td><td>Silicone isolator</td></tr><tr><td>ProLong Diamond Antifade Mountant</td><td>Thermo Fisher Scientific</td><td>P36965</td><td>Mounting medium</td></tr><tr><td>SEA BLOCK Blocking Buffer</td><td>Thermo Fisher Scientific</td><td>37527X3</td><td></td></tr><tr><td>Sodium azide</td><td>Sigma-Aldrich</td><td>S2002-100G</td><td></td></tr><tr><td>Tissue Path IV Tissue Cassettes</td><td>Thermo Fisher Scientific</td><td>22-272416</td><td></td></tr><tr><td>Triton &amp;Chi;-100</td><td>Sigma-Aldrich</td><td>X100</td><td>Generic term: octoxynol-9</td></tr><tr><td>Tube rotator and rotisseries</td><td>VWR</td><td>10136-084</td><td></td></tr><tr><td>Tween-20</td><td>Sigma-Aldrich</td><td>P1379</td><td>Generic term: Polysorbate 20</td></tr></tbody></table>

co-staining blood vessels and nerve fibers in adipose tissue

Recent studies have highlighted the critical role of angiogenesis and sympathetic innervation in adipose tissue remodeling during the development of obesity. Therefore, developing an easy and efficient method to document the dynamic changes in adipose tissue is necessary. Here, we describe a modified immunofluorescent approach that efficiently co-stains blood vessels and nerve fibers in adipose tissues. Compared to traditional and recently developed methods, our approach is relatively easy to follow and more efficient in labeling the blood vessels and nerve fibers with higher densities and less background. Moreover, the higher resolution of the images further allows us to accurately measure the area of the vessels, amount of branching, and length of the fibers by open source software. As a demonstration using our method, we show that brown adipose tissue (BAT) contains higher amounts of blood vessels and nerve fibers compared to white adipose tissue (WAT). We further find that among the WATs, subcutaneous WAT (sWAT) has more blood vessels and nerve fibers compared to epididymal WAT (eWAT). Our method thus provides a useful tool for investigating adipose tissue remodeling.

The distal region of the epididymal white adipose tissue (eWAT), medial region of the dorsolumbar subcutaneous white adipose tissue (sWAT), and medial region of the interscapular brown adipose tissue (BAT) were collected. The locations for collecting these tissues are indicated in Figure 1.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59266/59266fig1.jpg" /> 
Figure 1: Ana...

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Co-staining Blood Vessels and Nerve Fibers in Adipose Tissue

New blood vessel formation and sympathetic innervation play pivotal roles in adipose tissue remodeling. However, there remain technical issues in visualizing and quantitatively measuring adipose tissue. Here we present a protocol to successfully label and quantitatively compare the densities of blood vessels and nerve fibers in different adipose tissues.

Recent studies have highlighted the critical role of angiogenesis and sympathetic innervation in adipose tissue remodeling during the development of ...

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Immunology and Infection

9.1K Views.  University of Texas Health Science Center at Houston. Recente studies hebben gewezen op de cruciale rol van angiogenese en sympathische innovatie in Vetweefsel remodelleren tijdens de ontwikkeling van obesitas. Hier tonen we een gemodificeerde immunofluorescentiemethode die efficiënt bloedvaten en zenuwvezels in vetweefsel staat. Onze aanpak is rechttoe rechtaan en efficiënt zonder compromissen te sluiten over permanente efficiëntie en de kwaliteit.We verwerven de beelden met behulp van confocale microscopie. De hoge resolutie van de b...

Video: Co-staining Blood Vessels and Nerve Fibers in Adipose Tissue

Assessment of Human Adipose Tissue Microvascular Function Using Videomicroscopy

While obesity is closely linked to the development of metabolic and cardiovascular disease, little is known about mechanisms that govern these processes. It is hypothesized that pro-atherogenic mediators released from fat tissues particularly in association with central/visceral adiposity may promote pathogenic vascular changes locally and systemically, and the notion that cardiovascular disease may be the consequence of adipose tissue dysfunction continues to evolve. Here, we describe a unique method of videomicroscopy that involves analysis of vasodilator and vasoconstrictor responses of intact small human arterioles removed from the adipose depot of living human subjects. Videomicroscopy is used to examine functional properties of isolated microvessels in response to pharmacological or physiological stimuli using a pressured system that mimics in vivo conditions. The technique is a useful approach to gain understanding of the pathophysiology and molecular mechanisms that contribute to vascular dysfunction locally within the adipose tissue milieu. Moreover, abnormalities in the adipose tissue microvasculature have also been linked with systemic diseases. We applied this technique to examine depot-specific vascular responses in obese subjects. We assessed endothelium-dependent vasodilation to both increased flow and acetylcholine in adipose arterioles (50 - 350 &#181;m internal diameter, 2 - 3 mm in length) isolated from two different adipose depots during bariatric surgery from the same individual. We demonstrated that arterioles from visceral fat exhibit impaired endothelium-dependent vasodilation compared to vessels isolated from the subcutaneous depot. The findings suggest that the visceral microenvironment is associated with vascular endothelial dysfunction which may be relevant to clinical observation linking increased visceral adiposity to systemic disease mechanisms. The videomicroscopy technique can be used to examine vascular phenotypes from different fat depots as well as compare findings across individuals with different degrees of obesity and metabolic dysfunction. The method can also be used to examine vascular responses longitudinally in response to clinical interventions.

Videomicroscopy systems are used to examine functional properties of isolated adipose tissue arterioles in response to physiological and pharmacological stimuli. This technique can be used to examine microvascular phenotypes in different adipose tissue domains in obese humans.

While obesity is closely linked to the development of metabolic and cardiovascular disease, little is known about mechanisms that govern these processes. ...

Medicine

Quantitative Analysis of Cellular Composition in Advanced Atherosclerotic Lesions of Smooth Muscle Cell Lineage-Tracing Mice

Atherosclerosis remains the leading cause of death worldwide and, despite countless preclinical studies describing promising therapeutic targets, novel interventions have remained elusive. This is likely due, in part, to a reliance on preclinical prevention models investigating the effects of genetic manipulations or pharmacological treatments on atherosclerosis development rather than the established disease. Also, results of these studies are often confounding because of the use of superficial lesion analyses and a lack of characterization of lesion cell populations. To help overcome these translational hurdles, we propose an increased reliance on intervention models that employ investigation of changes in cellular composition at a single cell level by immunofluorescent staining and confocal microscopy. To this end, we describe a protocol for testing a putative therapeutic agent in a murine intervention model including a systematic approach for animal dissection, embedding, sectioning, staining, and quantification of brachiocephalic artery lesions. In addition, due to the phenotypic diversity of cells within late-stage atherosclerotic lesions, we describe the importance of using cell-specific, inducible lineage tracing mouse systems and how this can be leveraged for unbiased characterization of atherosclerotic lesion cell populations. Together, these strategies may assist vascular biologists to more accurately model therapeutic interventions and analyze atherosclerotic disease and will hopefully translate into a higher rate of success in clinical trials.

We propose a standardized protocol to characterize the cellular composition of late-stage murine atherosclerotic lesions including systematic methods of animal dissection, tissue embedding, sectioning, staining, and analysis of brachiocephalic arteries from atheroprone smooth muscle cell lineage tracing mice.

Atherosclerosis remains the leading cause of death worldwide and, despite countless preclinical studies describing promising therapeutic targets, novel ...

Lymphatic and Blood Network Analysis During Obesity

Lymphatic collecting vessels and lymph nodes are inevitably embedded in adipose tissue. The physiological significance of this observation remains still not elucidated. However, obesity is characterized by impaired lymphatic function and increased vessel permeability. Inversely, lymphatic dysfunction induces obesity in mice, suggesting a significant interplay between lymphatic vessels and the adipose tissue. Therefore, understanding factors leading to lymphatic dysfunction might open new therapeutic windows to prevent obesity and associated comorbidities. The first step in this process requires a precise and detailed visualization of the lymphatic network in healthy and inflamed adipose tissue. Here, we describe a rapid, inexpensive, and efficient method that allows to label and analyze lymphatic and blood vessels. This approach takes advantage of the skin-draining brachial lymph node localization within the subcutaneous adipose tissue. The lymphatic arborization of this tissue can be revealed by injecting fluorochrome-conjugated lectins subcutaneously. Moreover, the in vivo labeling approach provides a way to evaluate lymphatic vessel density and functions. Coupled to blood vessel, adipocyte and immune cell staining, the protocol allows for high-resolution mapping of the subcutaneous adipose tissue by 3D imaging.

Obesity is a growing global public health issue. It has been previously associated with lymphatic dysfunction, suggesting a vital crosstalk between the adipose tissue and the lymphatic system. Here, we propose an accessible methodology allowing the distinct labeling of blood and lymphatic vasculatures within the subcutaneous adipose tissue.

Lymphatic collecting vessels and lymph nodes are inevitably embedded in adipose tissue. The physiological significance of this observation remains still ...

Demonstrating Hairy and Glabrous Skin Innervation in a 3D Pattern Using Multiple Fluorescent Staining and Tissue Clearing Approaches

Skin innervation is an important part of the peripheral nervous system. Although the study of the cutaneous nerve fibers has progressed rapidly, most of the understanding of their distributional and chemical characteristics comes from conventional histochemical and immunohistochemical staining on thin tissue sections. With the development of the tissue clearing technique, it has become possible to view the cutaneous nerve fibers on thicker tissue sections. The present protocol describes multiple fluorescent staining on tissue sections at a thickness of 300 &#181;m from the plantar and dorsal skin of rat hindfoot, the two typical hairy and glabrous skin sites. Here, the calcitonin gene-related peptide labels the sensory nerve fibers, while phalloidin and lymphatic vessel endothelial hyaluronan receptor 1 label the blood and lymphatic vessels, respectively. Under a confocal microscope, the labeled sensory nerve fibers were followed completely at a longer distance, running in bundles in the deep cutaneous layer and freestyle in the superficial layer. These nerve fibers ran in parallel to or surrounded the blood vessels, and lymphatic vessels formed a three-dimensional (3D) network in the hairy and glabrous skin. The current protocol provides a more effective approach to studying skin innervation than the existing conventional methods from the methodology perspective.

The thickness of tissue sections limited the morphological study of the skin innervation. The present protocol describes a unique tissue clearing technique to visualize cutaneous nerve fibers in thick 300 &#181;m tissue sections under confocal microscopy.

Skin innervation is an important part of the peripheral nervous system. Although the study of the cutaneous nerve fibers has progressed rapidly, most of ...

Neuroscience

Coherent Anti-Stokes Raman Spectroscopy (CARS) Application for Imaging Myelination in Brain Slices

Coherent anti-Stokes Raman spectroscopy (CARS) is a technique classically employed by chemists and physicists to produce a coherent signal of signature vibrations of molecules. However, these vibrational signatures are also characteristic of molecules within anatomical tissue such as the brain, making it increasingly useful and applicable for Neuroscience applications. For example, CARS can measure lipids by specifically exciting chemical bonds within these molecules, allowing for quantification of different aspects of tissue, such as myelin involved in neurotransmission. In addition, compared to other techniques typically used to quantify myelin, CARS can also be set up to be compatible with immunofluorescent techniques, allowing for co-labeling with other markers such as sodium channels or other components of synaptic transmission. Myelination changes are an inherently important mechanism in demyelinating diseases such as multiple sclerosis or other neurological conditions such as Fragile X Syndrome or autism spectrum disorders is an emerging area of research. In conclusion, CARS can be utilized in innovative ways to answer pressing questions in Neuroscience and provide evidence for underlying mechanisms related to many different neurological conditions.

Coherent Anti-Stokes Raman Spectroscopy CARS Application for Imaging Myelination in Brain Slices

Visualizing myelination is an important goal for many researchers studying the nervous system. CARS is a technique that is compatible with immunofluorescence that can natively image lipids within tissue such as the brain illuminating specialized structures such as myelin.

Coherent anti-Stokes Raman spectroscopy (CARS) is a technique classically employed by chemists and physicists to produce a coherent signal of signature ...

Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte precursors, immune cells, fibroblasts, blood vessels, and nerve projections. Although the molecular control of cell type specification and how these cells interact have been increasingly delineated, a more comprehensive understanding of these adipose-resident cells can be achieved by visualizing their distribution and architecture throughout the whole tissue. Existing immunohistochemistry and immunofluorescence approaches to analyze adipose histology rely on thin paraffin-embedded sections. However, thin sections capture only a small portion of tissue; as a result, the conclusions can be biased by what portion of tissue is analyzed. We have therefore developed an adipose tissue clearing technique, Adipo-Clear, to permit comprehensive three-dimensional visualization of molecular and cellular patterns in whole adipose tissues. Adipo-Clear was adapted from iDISCO/iDISCO+, with specific modifications made to completely remove the lipid stored in the tissue while preserving native tissue morphology. In combination with light-sheet fluorescence microscopy, we demonstrate here the use of the Adipo-Clear method to obtain high-resolution volumetric images&#160;of an entire adipose tissue.

Due to the high lipid content, adipose tissue has been challenging to visualize using traditional histological methods. Adipo-Clear is a tissue clearing technique that allows robust labeling and high-resolution volumetric fluorescent imaging of adipose tissue. Here, we describe the methods for sample preparation, pretreatment, staining, clearing, and mounting for imaging.

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte ...

Biology

Visualization of 3D White Adipose Tissue Structure Using Whole-mount Staining

Adipose tissue is an important metabolic organ with high plasticity and is responsive to environmental stimuli and nutrient status. As such, various techniques have been developed to study the morphology and biology of adipose tissue. However, conventional visualization methods are limited to studying the tissue in 2D sections, failing to capture the 3D architecture of the whole organ. Here we present whole-mount staining, an immunohistochemistry method that preserves intact adipose tissue morphology with minimal processing steps. Hence, the structures of adipocytes and other cellular components are maintained without distortion, achieving the most representative 3D visualization of the tissue. In addition, whole-mount staining can be combined with lineage tracing methods to determine cell fate decisions. However, this technique has some limitations to providing accurate information regarding deeper parts of adipose tissue. To overcome this limitation, whole-mount staining can be further combined with tissue clearing techniques to remove the opaqueness of tissue and allow for complete visualization of entire adipose tissue anatomy using light-sheet fluorescent microscopy. Therefore, a higher resolution and more accurate representation of adipose tissue structures can be captured with the combination of these techniques.

The focus of the present study is to demonstrate the whole-mount immunostaining and visualization technique as an ideal method for 3D imaging of adipose tissue architecture and cellular component.

Adipose tissue is an important metabolic organ with high plasticity and is responsive to environmental stimuli and nutrient status. As such, various ...

Exploring Adipose Tissue Structure by Methylsalicylate Clearing and 3D Imaging

Obesity is a major worldwide public health issue that increases the risk to develop cardiovascular diseases, type-2 diabetes, and liver diseases. Obesity is characterized by an increase in adipose tissue (AT) mass due to adipocyte hyperplasia and/or hypertrophia, leading to profound remodeling of its three-dimensional structure. Indeed, the maximal capacity of AT to expand during obesity is pivotal to the development of obesity-associated pathologies. This AT expansion is an important homeostatic mechanism to enable adaptation to an excess of energy intake and to avoid deleterious lipid spillover to other metabolic organs, such as muscle and liver. Therefore, understanding the structural remodeling that leads to the failure of AT expansion is a fundamental question with high clinical applicability. In this article, we describe a simple and fast clearing method that is routinely used in our laboratory to explore the morphology of mouse and human white adipose tissue by fluorescent imaging. This optimized AT clearing method is easily performed in any standard laboratory equipped with a chemical hood, a temperature-controlled orbital shaker and a fluorescent microscope. Moreover, the chemical compounds used are readily available. Importantly, this method allows one to resolve the 3D AT structure by staining various markers to specifically visualize the adipocytes, the neuronal and vascular networks, and the innate and adaptive immune cells distribution.

Here, we describe a simple, inexpensive and fast clearing method to resolve the 3D structure of both mouse and human white adipose tissue using a combination of markers to visualize vasculature, nuclei, immune cells, neurons, and lipid-droplet coat proteins by fluorescent imaging.

Obesity is a major worldwide public health issue that increases the risk to develop cardiovascular diseases, type-2 diabetes, and liver diseases. Obesity ...

Human SATMVECs: Isolation and Adipocyte Cross-Talk Studies

Studying Adipose Endothelial Cell/Adipocyte Cross-Talk in Human Subcutaneous Adipose Tissue

Microvascular endothelial cells (MVECs) have many critical roles, including control of vascular tone, regulation of thrombosis, and angiogenesis. Significant heterogeneity in endothelial cell (EC) genotype and phenotype depends on their vascular bed and host disease state. The ability to isolate MVECs from tissue-specific vascular beds and individual patient groups offers the opportunity to directly compare MVEC function in different disease states. Here, using subcutaneous adipose tissue (SAT) taken at the time of insertion of cardiac implantable electronic devices (CIED), we describe a method for the isolation of a pure population of functional human subcutaneous adipose tissue MVEC (hSATMVEC) and an experimental model of hSATMVEC-adipocyte cross-talk.
hSATMVEC were isolated following enzymatic digestion of SAT by incubation with anti-CD31 antibody-coated magnetic beads and passage through magnetic columns. hSATMVEC were grown and passaged on gelatin-coated plates. Experiments used cells at passages 2-4. Cells maintained classic features of EC morphology until at least passage 5. Flow cytometric assessment showed 99.5% purity of isolated hSATMVEC, defined as CD31+/CD144+/CD45-. Isolated hSATMVEC from controls had a population doubling time of approximately 57 h, and active proliferation was confirmed using a cell proliferation imaging kit. Isolated hSATMVEC function was assessed using their response to insulin stimulation and angiogenic tube-forming potential. We then established an hSATMVEC-subcutaneous adipocyte co-culture model to study cellular cross-talk and demonstrated a downstream effect of hSATMVEC on adipocyte function.
hSATMVEC can be isolated from SAT taken at the time of CIED insertion and are of sufficient purity to both experimentally phenotype and study hSATMVEC-adipocyte cross-talk.

Author Spotlight: Insights into Cardiometabolic Diseases with Subcutaneous Adipose Tissue Microvasculature Studies 

Here, we describe a protocol for isolating, culturing, and phenotyping microvascular endothelial cells from human subcutaneous adipose tissue (hSATMVECs). Additionally, we describe an experimental model of hSATMVEC-adipocyte cross-talk.

Microvascular endothelial cells (MVECs) have many critical roles, including control of vascular tone, regulation of thrombosis, and angiogenesis. ...

Isolation and Identification of Vascular Endothelial Cells from Distinct Adipose Depots for Downstream Applications

Vascular endothelial cells lining the wall of the vascular system play important roles in a variety of physiological processes, including vascular tone regulation, barrier functions, and angiogenesis. Endothelial cell dysfunction is a hallmark predictor and major driver for the progression of severe cardiovascular diseases, yet the underlying mechanisms remain poorly understood. The ability to isolate and perform analyses on endothelial cells from various vascular beds in their native form will give insight into the processes of cardiovascular disease. This protocol presents the procedure for the dissection of mouse subcutaneous and mesenteric adipose tissues, followed by isolation of their respective arterial vasculature. The isolated arteries are then digested using a specific cocktail of digestive enzymes focused on liberating functionally viable endothelial cells. The digested tissue is assessed by flow cytometry analysis using CD31+/CD45&#8722; cells as markers for positive endothelial cell identification. Cells can be sorted for immediate downstream functional assays or used to generate primary cell lines. The technique of isolating and digesting arteries from different vascular beds will provide options for researchers to evaluate freshly isolated vascular cells&#160;from arteries of interest and allow them to perform a wide range of functional tests on specific cell types.

This protocol details a method for the dissection of mouse adipose depots and the isolation and digestion of respective arteries to liberate and then identify the endothelial cell population. Freshly isolated cells used in downstream applications will advance the understanding of vascular cell biology and the mechanisms of vascular dysfunction.

Vascular endothelial cells lining the wall of the vascular system play important roles in a variety of physiological processes, including vascular tone ...

Co-staining Blood Vessels and Nerve Fibers in Adipose Tissue

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