Name: co-staining blood vessels and nerve fibers in adipose tissue
Uploaded: 2019-02-13T15:00:00
Description: Watch this Scientific Journal Video about Co-staining Blood Vessels and Nerve Fibers in Adipose Tissue at JoVE.com

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 achi...

<ol>
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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose+tissue+remodeling+and+obesity.">Adipose tissue remodeling and obesity.</a> Journal of Clinical Investigations. 121 (6), 2094-2101 (2011).</li><li>Sun, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endotrophin+triggers+adipose+tissue+fibrosis+and+metabolic+dysfunction.">Endotrophin triggers adipose tissue fibrosis and metabolic dysfunction.</a> Nature Communication. 5, 3485 (2014).</li><li>Zhao, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Divergent+functions+of+endotrophin+on+different+cell+populations+in+adipose+tissue.">Divergent functions of endotrophin on different cell populations in adipose tissue.</a> American Journal of Physiology-Endocrinology and Metabolism. 311 (6), E952-E963 (2016).</li><li>Zhao, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+Overexpression+of+VEGF-A+in+Adipose+Tissue+Promotes+Energy+Expenditure+via+Activation+of+the+Sympathetic+Nervous+System.">Transient Overexpression of VEGF-A in Adipose Tissue Promotes Energy Expenditure via Activation of the Sympathetic Nervous System.</a> Molecular and Cellular Biology. , (2018).</li><li>Xue, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia-independent+angiogenesis+in+adipose+tissues+during+cold+acclimation.">Hypoxia-independent angiogenesis in adipose tissues during cold acclimation.</a> Cell Metabolism. 9 (1), 99-109 (2009).</li><li>Chen, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LncRNA+TDRG1+enhances+tumorigenicity+in+endometrial+carcinoma+by+binding+and+targeting+VEGF-A+protein.">LncRNA TDRG1 enhances tumorigenicity in endometrial carcinoma by binding and targeting VEGF-A protein.</a> BBA Molecular Basis of Disease. 1864 (9 Pt B), 3013-3021 (2018).</li><li>Sun, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dichotomous+effects+of+VEGF-A+on+adipose+tissue+dysfunction.">Dichotomous effects of VEGF-A on adipose tissue dysfunction.</a> Proceedings of the National Academy of Sciences of the United States of America. 109 (15), 5874-5879 (2012).</li><li>During, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose+VEGF+Links+the+White-to-Brown+Fat+Switch+With+Environmental,+Genetic,+and+Pharmacological+Stimuli+in+Male+Mice.">Adipose VEGF Links the White-to-Brown Fat Switch With Environmental, Genetic, and Pharmacological Stimuli in Male Mice.</a> Endocrinology. 156 (6), 2059-2073 (2015).</li><li>Elias, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose+tissue+overexpression+of+vascular+endothelial+growth+factor+protects+against+diet-induced+obesity+and+insulin+resistance.">Adipose tissue overexpression of vascular endothelial growth factor protects against diet-induced obesity and insulin resistance.</a> Diabetes. 61 (7), 1801-1813 (2012).</li><li>Sung, H. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose+vascular+endothelial+growth+factor+regulates+metabolic+homeostasis+through+angiogenesis.">Adipose vascular endothelial growth factor regulates metabolic homeostasis through angiogenesis.</a> Cell Metabolism. 17 (1), 61-72 (2013).</li><li>Cao, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiogenesis+and+vascular+functions+in+modulation+of+obesity,+adipose+metabolism,+and+insulin+sensitivity.">Angiogenesis and vascular functions in modulation of obesity, adipose metabolism, and insulin sensitivity.</a> Cell Metabolism. 18 (4), 478-489 (2013).</li><li>Sun, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brown+adipose+tissue+derived+VEGF-A+modulates+cold+tolerance+and+energy+expenditure.">Brown adipose tissue derived VEGF-A modulates cold tolerance and energy expenditure.</a> Molecular Metabolism. 3 (4), 474-483 (2014).</li><li>Zeng, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sympathetic+neuro-adipose+connections+mediate+leptin-driven+lipolysis.">Sympathetic neuro-adipose connections mediate leptin-driven lipolysis.</a> Cell. 163 (1), 84-94 (2015).</li><li>Gage, G. J., Kipke, D. R., Shain, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+animal+perfusion+fixation+for+rodents.">Whole animal perfusion fixation for rodents.</a> Journal of Visualized Experiments. (65), (2012).</li><li>Berry, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+of+adipose+tissue.">Imaging of adipose tissue.</a> Methods in Enzymology. 537, 47-73 (2014).</li><li>Jiang, H., Ding, X., Cao, Y., Wang, H., Zeng, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dense+Intra-adipose+Sympathetic+Arborizations+Are+Essential+for+Cold-Induced+Beiging+of+Mouse+White+Adipose+Tissue.">Dense Intra-adipose Sympathetic Arborizations Are Essential for Cold-Induced Beiging of Mouse White Adipose Tissue.</a> Cell Metabolism. 26 (4), 686-692 (2017).</li><li>Chi, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-Dimensional+Adipose+Tissue+Imaging+Reveals+Regional+Variation+in+Beige+Fat+Biogenesis+and+PRDM16-Dependent+Sympathetic+Neurite+Density.">Three-Dimensional Adipose Tissue Imaging Reveals Regional Variation in Beige Fat Biogenesis and PRDM16-Dependent Sympathetic Neurite Density.</a> Cell Metabolism. 27 (1), 226-236 (2018).</li><li>Zudaire, E., Gambardella, L., Kurcz, C., Vermeren, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+computational+tool+for+quantitative+analysis+of+vascular+networks.">A computational tool for quantitative analysis of vascular networks.</a> PLoS One. 6 (11), e27385 (2011).</li><li>An, Y. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiopoietin-2+in+white+adipose+tissue+improves+metabolic+homeostasis+through+enhanced+angiogenesis.">Angiopoietin-2 in white adipose tissue improves metabolic homeostasis through enhanced angiogenesis.</a> eLife. 6, (2017).</li><li>Chi, J., Crane, A., Wu, Z., Cohen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipo-Clear:+A+Tissue+Clearing+Method+for+Three-Dimensional+Imaging+of+Adipose+Tissue.">Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue.</a> Journal of Visualized Experiments. (137), (2018).</li></ol>

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 
			https://ccrod.cancer.gov/confluence/display/ROB2/Home</td>
		</tr>
		<tr>
			<td>Anti-mouse endomucin antibody</td>
			<td>R&#38;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=0.17mm</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 &#935;-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...

Watch this Scientific Journal Video about Co-staining Blood Vessels and Nerve Fibers in Adipose Tissue at JoVE.com

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 ...

Recent studies have highlighted the critical role of angiogenesis and sympathetic innovation in Adipose tissue remodeling during the development of obesity. Here we demonstrate a modified immunofluorescence method that efficiently stands blood vessels and nerve fibres in Adipose tissue. Our approach is straight forward and efficient with no compromise on standing efficiency and the quality.We acquire the images using confocal microscopy. The high resolution of the images allows us to reconstruct the blood vessel networks and accurately analyse its characteristics. Begin this procedure with anesthesia and perfusion of six week C57 Black-6J male mice as detailed in the text protocol.Dissect white and brown adipose issues from the mice. Use scissors to break the tissue samples into small pieces of chunk. Transfer the small chunks with the sterilized tweezers into the tissue embedding cassettes with proper labeling of the tissue samples on the cassettes.Immerse the embedded cassettes containing tissue samples into the fixation solution at four degrees celsius for 24 to 48 hours. Transfer the small chunks was sterilized tweezers into a Petri Dish. Wash the samples 3 times with brush 1x PBS buffer at 0.5 milliliters per wash.Now cut the fixed tissue samples into approximately two cubic millimeter cubes with the sterilized scissors. For permeabalisation, transfer the samples into a 1.5 milliliter tube containing one milliliter of 1%Triton PBS buffer and gently rotate the tubes at room temperature for 1 hour at 18 RPM. After an hour carefully remove the 1%Triton PBS buffer by aspiration wash the samples three times by adding the 1x PBS directly into the same tubes.During each wash, invert the tubes several times. For blocking, add 0.5 milliliter of blocking buffer to the samples and incubator at room temperature for two hours with gentle rotation. To prepare 0.4 milliliters of primary antibody solution, dilute two microliters of anti Endomusin and two microliters of anti-tyrosine hydroxy lase antibodies until 396 microliters of blocking buffer.Vortex and spin down to recover the volume. Now, carefully remove the blocking buffer from the tissue chunks. Add 100 microliters of the prepared primary antibodies solution into tubes and incubate at four degrees celcius overnight.The next day, carefully collect the primary antibody solution for reuse if desired. Wash the samples three times with 1x PBST at 500 micro litres per wash for 30 minutes with gentle rotation at 18 RPM. For the preparation of secondary antibodies solution, dilute two microlitres of flouro-flouro conjugated anti-god IGG in two microlitres of flouro-flouro conjugated anti-rabid IGG into 396 microliters of blocking buffer.Vortex and spin briefly to collect all the liquid. Now, remove the last wash buffer from the samples. Add 100 microliters of secondary antibody solution into tubes.Incubate the samples at room temperature for two hours with gentle rotation at 18 RPM. After carefully removing the secondary antibody solution, wash the samples three times with 1x PBST at 500 microlitres per wash for 30 minutes each with gentle rotation at 18 RPM. For optical clearance, immerse the samples in one milliliter of 90%Glycerol and keep the samples at four degrees celcius in darkness until they become transparent.Add here a silicon isolator to the slide to create a well for volume imaging. Keep the height of the silicone to or slightly less than that of the sample. Carefully transfer the samples into the well and fill it with the mounting medium.Lay a coverslip on the surface and seal the quarters of the coverslip with high viscosity medium. Let the mounting medium cure for 24 hours at room temperature and darkness. After curing is complete, fully seal the edges of the coverslip for optimum sample longevity.Acquire Z stack images with the 20 times objective of a confocal microscope and its corresponding software. Start the system. Click on the configuration tab and activate both the Argon laser and the He-Ne 633 laser.Click on the acquire tab. In the visible laser lines panel, move the corresponding intensity sliders up to choose the laser lines up 488 nanometers and 633 nanometers. Initially the intensities can be set to 20 to 30%Now select the 488, 561, 633 triple dichroic mirror.Activate the photo multipliers by clicking on the active check boxes. Choose photomultiplier one for the emission exerted by the 488 nanometer laser and photomultiplier 34 for that of the 633 nanometer laser. Set the wavelength range to between 500 and 550 nanometers for photo multiplier one.In 650 to 750 nanometers, for photo multiplier three. Select the pseudo color to be used for image display by double clicking the colored rectangle beside each photo multiplier and choosing a color from the pop-up menu. Now click on live to check the image at the samples.Use the Z position nob to select a plane of focus within the XY region of interest. Adjust the brightness of the images by turning the laser intensity, smart gain, and offset. For each fluorescence channel, perform this adjustment under the quick lookup table display mode.Click on the quick look up table button to enter the quick lookup table mode in which saturated pixels are displayed in blue to aid the setting of appropriate brightness level. Click twice on the quick look up table button to go back to the pseudo color display mode. While scanning, turn the Z position nob counter clockwise to move the plane of focus to one end of the volume of interest.Then click on the end arrowhead to set the scanning and position. Turn the Z position nob of clockwise to move the plane of focus through the specimen to the other end of the volume of interest. Then click on the begin arrowhead to set the scanning begin position.Adjust the Z step size to three microns. Change the image quality by selecting a format of 1024 by 1024, A speed of 100 Hz, and a line average of two. Select start to initiate the Z stack image acquisition For maximum projection of the acquired image stack, Click on the process tab and then tool;select 3D projection and enter maximum in the method list without changes to X, Y and Z.Set threshold to zero, click on apply.The maximum intensity of the Z volume will be stacked into a 2D image which is displayed. To analyse the 2D images via open source software, open the stacked images in the software. Adjust vessel diameter and intensity until all vessel structures can be properly selected.Select run analysis and export the data following guidance of the software. To analyse the 3D images via the licence software, open the raw data of images with the software. Adjust the intensity threshold and other parameters until the vessel structures in each layer of the Z volume are properly segmented.Skeletonize the resulting binarised image stack to obtain a special graph. Analyse the selected segment characteristics and export the data following the guidance of the software. The distal region epididymal white adipose tissue, the medial region of the dorsal lumbar subcutaneous white adipose tissue and the medial region of the intra-scapular brown adipose tissue were collected.After a whole mount staining, the tissue trunks were mounted and imaged with the confocal microscope. More blood vessels were positively stained. With the anti-Endomusin antibody in the glycerol incubated subcutaneous white adipose tissue, suggesting that the clearing step is critical for complete staining of the blood vessels.To determine whether blood vessels and nerve fibers exhibit different patterns among the depos with this method, Communal fluorescence staining was performed in adipose tissue with antibodies with anti-Endomusin to show blood vessels and with anti-tyrosine hydroxy lase to show nerve fibres. Interestingly, results show that they were significantly more blood vessels than nerve fibres in brown adipose tissue as compared to white adipose tissue. Among the white adipose tissue, the subcutaneous white adipose tissue exhibited higher blood vessel density than the epididymal white adipose tissue.Of note, while the nerve fibres expanded in parallel with the blood vessels, they did not show significant co-localization. The vessel area, number of junctions, and tube length were then qualitatively measured with the 2D method in these three types of adipose tissue and found similar results. This method efficiently cos-dens blood vessels and nerve fibers and adipose tissue.It sounds as a useful tool for studying the dynamic change in adipose tissue during the development of obesity. Powerful mouth hy-de is toxic. Please carry out P-ifa involved steps in a A wider skin contact and and inhalation and properly handle the P-ifa Because the laser beams have become focal microscope yet harm the eye.Avoid eye exposure to the beams coming from the objective,

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