T. S. is supported by grants from the DFG German Research Foundation (332853055), Else Kr&#246;ner-Fresenius-Stiftung (2015_A197), and the Medical Faculty of the RWTH Aachen (RWTH Returner Program). V. G. P. is supported by research fellowships from Deutsche Gesellschaft fur Nephrologie, the Alexander von Humboldt Foundation, and the National Health and Medical Research Council of Australia. D. H. E is supported by Fondation LeDucq. R. K. is supported by grants from the DFG (KR-4073/3-1, SCHN1188/5-1, SFB/TRR57, SFB/TRR219), the State of Northrhinewestfalia (MIWF-NRW) and the Interdisciplinary Centre for Clinical Research at RWTH Aachen University (O3-11).

NOTE: All experimental procedures described here were approved by the Institutional Animal Care and Use Committee (IACUC) of Oregon Health and Science University, Portland, Oregon, USA, and relevant local authorities in Aachen, Germany.
1. Retrograde Abdominal Aortic Perfusion and Fixation of Mouse Kidneys
<ol>
	<li>Prepare solutions the same day or evening before and store in a fridge overnight. Warm solutions to room temperature (RT) before using.</li>
	<li>Make a ...

<ol>
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The authors have nothing to disclose.

Optical clearing techniques have received wide attention for 3-D visualization and quantification of microanatomy in various organs. Here, solvent-based clearing method (ECi) was combined with immunolabeling for 3-D imaging of whole tubules in kidney slices. This method is simple, inexpensive, and quick. However, other research questions may be best answered with other clearing protocols5. It is also important to keep in mind that solvent-based methods cause tissue-shrinkage at variable degrees, m...

The growing interest in studying entire organs or large multicellular structures have led to the development of optical clearing methods that involve imaging of transparent tissue in three dimensions. Until recently, the best methods to estimate cell number, length, or volume of whole structures have been stereology or exhaustive serial sectioning, which is based on the systemic sampling of tissue for subsequent analysis in two dimensions1,2,3. However, these methods are time-consuming and need a high level of training and expertise4. Optical clearing methods overcome these problems by equilibrating the refractive index throughout a sample to make tissue translucent for 3-D imaging5,6,7.
Several optical clearing methods have been developed which fall into two main categories: solvent-based and aqueous-based methods. Aqueous-based methods can be further divided into simple immersion8,9, hyperhydration10,11, and hydrogel embedding12,13. Solvent-based methods dehydrate the tissue, remove lipids and normalize the refractive index to a value around 1.55. Limitations of most solvent-based methods are quenching of endogenous fluorescence of commonly used reporter proteins such as GFP, solvent toxicity, capacity to dissolve glues used in some imaging chambers or objective lenses, and shrinkage of tissue during dehydration14,15,16,17,18,19,20,21. However, solvent-based methods are simple, time-efficient, and can work in a number of different tissue types.
Aqueous-based methods rely on the immersion of the tissue in aqueous solutions that have refractive indices in the range of 1.38-1.528,11,12,22,23,24. These methods were developed to preserve endogenous fluorescent reporter protein emission and prevent dehydration-induced shrinkage, but limitations of most aqueous-based clearing methods include a longer duration of the protocol, tissue expansion, and protein modification (i.e. partial denaturation of proteins by urea in hyperhydrating protocols such as ScaleA2)7,11,23,25. ScaleS addressed tissue expansion by combining urea with sorbitol, which counterbalances by dehydration the urea-induced tissue expansion, and preserved the tissue ultrastructure as evaluated by electron microscopy10. Tissue shrinkage or expansion affects the absolute sizes of structures, distances between objects, or cell density per volume; thus, the measurement of size changes upon clearing of the tissue may help interpret the obtained results7,26.
In general, a protocol for optical clearing consists of multiple steps, including pretreatment, permeabilization, immunolabeling (if required), refractive index matching, and imaging with advanced light microscopy (e.g., two-photon, confocal, or light-sheet fluorescence microscopy). Most of the clearing approaches have been developed to visualize neuronal tissue, and emerging studies have validated their application in other organs5. This comprehensive tool has been previously demonstrated to allow reliable and efficient analysis of kidney structures, including glomeruli27,28, immune infiltrates28, vasculature28, and tubule segments29, and it is an ideal approach to better the understanding of glomerular function and tubule remodeling in health and disease.
Summarized here is a solvent-based method that combines immunostaining of kidney tubules; optical clearing with cheap, non-toxic, and ready-to-use chemical ethyl cinnamate (ECi); and confocal microscopy imaging that allows complete tubule visualization and quantification. This method is simple, combines antigen-retrieval of kidney slices with staining of commercial antibodies, and does not require specialized equipment, which makes it accessible to most laboratories.

<table>
	<tbody>
		<tr>
			<td>0.22 &#181;m filter</td>
			<td>Fisher Scientific</td>
			<td>09-761-112</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL conical tube</td>
			<td>Fisher Scientific</td>
			<td>339650</td>
			<td></td>
		</tr>
		<tr>
			<td>21 gauge butterfly needle</td>
			<td>Braun</td>
			<td>Venofix</td>
			<td></td>
		</tr>
		<tr>
			<td>3-way stopcock</td>
			<td>Fisher Scientific</td>
			<td>K420163-4503</td>
			<td></td>
		</tr>
		<tr>
			<td>3D analyis software</td>
			<td>Bitplane AG</td>
			<td>IMARIS</td>
			<td></td>
		</tr>
		<tr>
			<td>3D analyis software</td>
			<td>Cellprofiler</td>
			<td></td>
			<td>free open-source software</td>
		</tr>
		<tr>
			<td>5-0 silk suture</td>
			<td>Fine Science Tools</td>
			<td>18020-50</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml plastic syringes</td>
			<td>Fisher Scientific</td>
			<td>14-817-57</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-BrdU monoclonal antibody</td>
			<td>Roche</td>
			<td>11296736001</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody diluent</td>
			<td>Dako</td>
			<td>S0809</td>
			<td></td>
		</tr>
		<tr>
			<td>CD31-647</td>
			<td>BioLegend</td>
			<td>102516</td>
			<td></td>
		</tr>
		<tr>
			<td>Citrate-based antigen retrieval solution</td>
			<td>Vector Laboratories</td>
			<td>H-3300</td>
			<td></td>
		</tr>
		<tr>
			<td>curved hemostat</td>
			<td>Fisher Scientific</td>
			<td>13-812-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Dako Wash Buffer</td>
			<td>Agilent</td>
			<td>S3006</td>
			<td></td>
		</tr>
		<tr>
			<td>dissecting microscope</td>
			<td>Motic</td>
			<td>DSK-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Embedding cassettes</td>
			<td>Carl Roth</td>
			<td>E478.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Merck</td>
			<td>100983</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl cinnamate</td>
			<td>Sigma-Aldrich</td>
			<td>112372</td>
			<td></td>
		</tr>
		<tr>
			<td>Flexible film/Parafilm M</td>
			<td>Sigma-Aldrich</td>
			<td>P7793</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-AQP2</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-9882</td>
			<td></td>
		</tr>
		<tr>
			<td>Guinea pig anti-NKCC2</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>DOI: 10.1681/ASN.2012040404</td>
		</tr>
		<tr>
			<td>HCl</td>
			<td>Carl Roth</td>
			<td>P074.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sagent Pharmaceuticals</td>
			<td>401-02</td>
			<td></td>
		</tr>
		<tr>
			<td>hemostat</td>
			<td>Agnthos</td>
			<td>312-471-140</td>
			<td></td>
		</tr>
		<tr>
			<td>horizontal rocker</td>
			<td>Labnet</td>
			<td>S2035-E</td>
			<td></td>
		</tr>
		<tr>
			<td>Imaging dish</td>
			<td>Ibidi</td>
			<td>81218</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>MWI Animal Health</td>
			<td>501090</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro serrefine</td>
			<td>Fine Science Tools</td>
			<td>18052-03</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Fisher Scientific</td>
			<td>S318-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Operating scissors</td>
			<td>Merit</td>
			<td>97-272</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Thermo Fischer Scientific</td>
			<td>O4042-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-phoshoThr53-NCC</td>
			<td>PhosphoSolutions</td>
			<td>p1311-53</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicone elastomer</td>
			<td>World Precision Instruments Kwik-Sil</td>
			<td>KWIK-SIL</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>Sigma-Aldrich</td>
			<td>S2002</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue slicer</td>
			<td>Zivic Instruments</td>
			<td>HSRA001-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Acros Organics</td>
			<td>AC215682500</td>
			<td></td>
		</tr>
		<tr>
			<td>Vannas scissors</td>
			<td>Fine Science Tools</td>
			<td>15000-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Vibratome</td>
			<td>Lancer</td>
			<td>Series 1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>MWI Animal Health</td>
			<td>AnaSed Inj SA (Xylazine)</td>
			<td></td>
		</tr>
	</tbody>
</table>

optical clearing and imaging of immunolabeled kidney tissue

Optical clearing techniques render tissue transparent by equilibrating the refractive index throughout a sample for subsequent three-dimensional (3-D) imaging. They have received great attention in all research areas for the potential to analyze microscopic multicellular structures that extend over macroscopic distances. Given that kidney tubules, vasculature, nerves, and glomeruli extend in many directions, which have been only partially captured by traditional two-dimensional techniques so far, tissue clearing also opened up many new areas of kidney research. The list of optical clearing methods is rapidly growing, but it remains difficult for beginners in this field to choose the best method for a given research question. Provided here is a simple method that combines antibody labeling of thick mouse kidney slices; optical clearing with cheap, non-toxic and ready-to-use chemical ethyl cinnamate; and confocal imaging. This protocol describes how to perfuse kidneys and use an antigen-retrieval step to increase antibody- binding without requiring specialized equipment. Its application is presented in imaging different multicellular structures within the kidney, and how to troubleshoot poor antibody penetration into tissue is addressed. We also discuss the potential difficulties of imaging endogenous fluorophores and acquiring very large samples and how to overcome them. This simple protocol provides an easy-to-setup and comprehensive tool to study tissue in three dimensions.

Kidneys are complex organs comprised of 43 different cell types31. Most of these cells form large multicellular structures such as glomeruli and tubules, and their function is highly dependent on interactions with each other. Classical 2-D histological techniques partially capture these large structures and may miss focal changes within intact structures31. Thus, 3-D analysis using optical clearing techniques helps to understand how they function in health and disease.
...

Watch this Scientific Journal Video about Optical Clearing and Imaging of Immunolabeled Kidney Tissue at JoVE.com

Optical Clearing and Imaging of Immunolabeled Kidney Tissue

The combination of antibody labeling, optical clearing, and advanced light microscopy allows three-dimensional analysis of complete structures or organs. Described here is a simple method to combine immunolabeling of thick kidney slices, optical clearing with ethyl cinnamate, and confocal imaging that enables visualization and quantification of three-dimensional kidney structures.

Optical clearing techniques render tissue transparent by equilibrating the refractive index throughout a sample for subsequent three-dimensional (3-D) ...

Growing interest in studying large, multi-cellular structures led to the development of this optic clearing technique which allows for a three-dimensional visualization and quantification of large structures within transparent organs. This protocol provides a simple, cheap, easy to set up and comprehensive tool to investigate tissue in three dimensions. Multi-layered nature of a disease cannot always be simulated by traditional two-dimensional techniques.Three-dimensional analysis of tissue can help to better diagnose and monitor disease. This method is not restricted to kidney tissue and can be applied to any normal and diseased tissue. This simple protocol is easy to follow for any scientist with some experience in animal handling.It is important to test different antibodies in case of poor antibody labeling, and conduct secondary antibody only controls. Start by preparing heparin containing PBS and 3.0%paraformaldehyde in 1X PBS according to the manuscript directions and transferring the solutions into separate 50 mL plastic syringes. Paraformaldehyde is toxic and should be prepared in the fume hood.Collect and profuse the kidney according to the manuscript directions. Then, remove the capsule and cut the kidney into one mm thick coronal slices. Immerse the kidney slices with the prepared paraformaldehyde solution in a 15 mL conical tube.After fixation, wash the kidney slice twice with 1X wash buffer for one hour on a horizontal rocker and then proceed with antigen retrieval. Heat up 300 mL of 1X antigen unmasking solution in a 500 mL beaker. Then, enclose a kidney slice in an embedding cassette permeable to the heated buffer and stir it for one hour at 92 to 98 degrees Celsius.It is important to keep the temperature stable between 92 and 98 degrees Celsius for this antigen retrieval step. Then transfer the slice into 10 mL of 1X wash buffer with 0.1%Triton X-100 and rock it overnight. The next day, wash the slice twice with 10 mL of fresh 1X wash buffer for one hour.Dilute the primary antibody in 500 microliters of normal antibody diluent. Gently rock the kidney slice in diluted primary antibody for four days at 37 degrees Celsius. After the incubation, wash the kidney slice in 10 mL of 1X wash buffer overnight at room temperature, changing the buffer once after eight hours.Dilute the secondary antibodies in 500 microliters of normal antibody diluent and incubate with the kidney slice for four days at 37 degrees Celsius. Please note that from this step forward, the kidney slice should be protected from light. After incubation with secondary antibodies, wash the kidney slice in 10 mL of 1X wash buffer overnight at room temperature.To dehydrate the kidney slice, transfer it to five mL of high grade 100%ethanol and rock it for two hours at room temperature, refreshing the ethanol after one hour. Immerse the kidney slice in two mL of ethyl cinnamate and rock gently overnight. When ready to image the tissue, add 600 to 1000 microliters of ethyl cinnamate into a glass-bottom dish and transfer the kidney slice into the dish.Place a round cover slip on the kidney slice and seal the dish with paraffin film. Place the dish onto the microscope imaging platform and image the tissue according to the manuscript directions. This protocol has been used to successfully perform 3D analysis on kidney tissue and evaluate cell functions and interactions.The method may cause fluorescent reporter quenching but other strong fluorescent proteins may resist signal quenching. Abdominal aortic profusion can improve antibody diffusion. In some instances, poor antibody penetration results in a superficial signal.This can be corrected with higher concentrations or replenishment of antibody. Intravascular delivery should be considered if the antigen of interest is expressed in kidney vasculature. However, antibodies targeting proteins in the apical membrane of tubule epithelial cells do not cross the glomerular filtration barrier and cause unspecific signals.The tissue can be labeled with the antibodies to detect a specific cell population or to visualize whole tubule segments. Furthermore, colocalization of proteins in 3D can be achieved by combining multiple antibodies. It is important to remember that the antibody labeling can be improved with good kidney profusion, the use of antigen retrieval step, and high antibody concentration.Some strong reporter proteins may resist quenching of fluorescent signal following this protocol. To test this, skip the antigen retrieval and antibody labeling steps. This technique respects the three-dimensional nature of structures and opens up many new areas for research.It eliminates a required assumptions and interferences associated with traditional two-dimensional analysis.

optical-clearing-and-imaging-of-immunolabeled-kidney-tissue

Research

JoVE Journal

Medicine

10.3K ビュー.  University Hospital RWTH Aachen. 大規模な多細胞構造の研究に対する関心の高まりは、透明臓器内の大きな構造の3次元可視化と定量化を可能にするこの光学クリアリング技術の開発につながった。このプロトコルは、簡単で安価で簡単に設定でき、3次元で組織を調査するための包括的なツールを提供します。疾患の多層的性質は、常に伝統的な2次元技術によってシミュレートされるとは限りません。