Name: optical clearing and imaging of immunolabeled kidney tissue
Uploaded: 2019-07-22T14:00:00
Description: Watch this Scientific Journal Video about Optical Clearing and Imaging of Immunolabeled Kidney Tissue at JoVE.com

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
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	<li>Prepare solutions the same day or evening before and store in a fridge overnight. Warm solutions to room temperature (RT) before using.</li>
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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) ...

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