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 fresh batch of 3% paraformaldehyde (PFA) in 1x phosphate-buffered saline (PBS). About 50-100 mL PFA is needed per mouse.
	<ol>
		<li>To make 1 L of 3% PFA: Weigh 30 g of PFA and add 800 mL of distilled water in the fume hood. Stir and heat to 50-60 &#176;C. Do not heat above 70 &#176;C. 
		CAUTION: PFA is toxic. Prepare PFA in the fume hood.</li>
		<li>Slowly add several drops of 2N NaOH. Wait a few min until PFA goes into the solution or add a few more drops. Some small chunks will not dissolve.</li>
		<li>Remove the solution from heat. Add 50 mL of 20x PBS. Chill on ice to RT.</li>
		<li>Adjust the pH to 7.3-7.4 with HCl. Add distilled water to 1 L.</li>
		<li>Remove any undissolved particles by filtering 1x PBS and 3% PFA with 0.22 &#181;m filter.</li>
	</ol>
	</li>
	<li>Add 1,000 units of Heparin to 1 L of 1x PBS. Transfer 1x PBS containing heparin and 3% PFA in 1x PBS into separate 50 mL plastic syringes. 
	NOTE: If available, pressure-controlled pump set at 80-100 mmHg or hydrostatic pressure (drip method, the height of the perfusion solutions: 160-200 cm above animal) can be used for kidney perfusion.</li>
	<li>Connect the PBS, PFA and a blunted 21 G butterfly needle to a three-way stopcock. Make sure there are no air bubbles in the whole system.</li>
	<li>Label a 15 mL conical tube and dispense 10 mL of PFA into it.</li>
	<li>Deeply anesthetize a male or female C57BL/6 mouse 12-24 weeks of age using 120 mg/kg bodyweight ketamine and 16 mg/kg bodyweight xylazine. The animal must be checked for complete absence of responsiveness by pinching the reflexes before proceeding to surgery (e.g., toe pinch reflex).</li>
	<li>Once the animal has reached a surgical plane of anesthesia, place it on its back under the dissecting microscope. Surgically open the abdomen with a midline abdominal incision using an operating scissors and expose the abdominal aorta.</li>
	<li>Clamp the abdominal aorta right above the branching to the iliac artery with a curved hemostat. Then clamp the abdominal aorta right below the renal arteries with a micro serrefine. Make a small incision (1 mm) on the abdominal aorta between the two clamps with vannas scissors. Insert the butterfly needle into the incision slowly and be careful not to rip the abdominal aorta open.</li>
	<li>Ligate the right renal artery with an 5-0 silk suture and remove the right kidney for other analysis if only one kidney is needed for perfusion and fixation.</li>
	<li>Remove the micro serrefine, transect the portal vein with vannas scissors, and immediately perfuse with 50 mL of PBS containing heparin, then switch and perfuse with 50 mL of 3% PFA. 
	NOTE: High perfusion pressure through abdominal aorta is required to open renal tubules for better antibody diffusion through tissue. Perfusion through heart may not open renal tubules.</li>
	<li>Collect the perfused kidney carefully and avoid puncturing or squeezing the tissue.</li>
	<li>Remove the capsule and cut the kidney into 1 mm thick coronal slices. Use a slicer matrix (Table of Materials) to standardize slice thickness. 
	NOTE: Alternatively, consider using a vibratome.</li>
	<li>Immerse the kidney slices with the prepared PFA in the labeled 15 mL conical tube.</li>
	<li>Dispense 10 mL of PFA to another labeled 15 mL conical tube. Flush the three-way stopcock and butterfly needle with PBS before moving to the next mouse.</li>
	<li>Carry out post-fixation overnight at RT protected from light.</li>
</ol>
2. Tissue Preparation and Immunostaining
<ol>
	<li>After post-fixation, wash the kidney slice twice with 1x wash buffer (Table of Materials) for 1 h on a horizontal rocker at RT.</li>
	<li>Perform antigen retrieval. Heat up 300 mL of 1x antigen unmasking solution (Table of Materials) in a 500 mL beaker to 92-98 &#176;C. Enclose the slice in embedding cassette permeable to the heated buffer with stirring for 1 h at 92-98 &#176;C. Remove the beaker from heat and leave it to cool to RT. 
	NOTE: Some vendors test their antibodies for immunohistochemistry application and will include a suggested antigen retrieval method in the datasheet. Therefore, some epitopes may require a more basic buffer (e.g., pH 9).</li>
	<li>Transfer the slice into 10 mL of 1x wash buffer with 0.1% Triton X-100 and rock overnight at RT. Wash the slices 2x with 10 mL of fresh 1x wash buffer for 1 h the next day.</li>
	<li>Dilute the primary antibody in 500 &#181;L of normal antibody diluent (Table of Materials). Begin with a concentration of &#173;&#173;&#173;1:50-1:100. Gently rock the kidney slice in diluted primary antibody for 4 d at 37 &#176;C. 
	NOTE: Since each antibody has unique properties, temperature during antibody incubation and dilutions of antibody need to be optimized for individual probes. For secondary antibody-only controls, incubate kidney tissue in diluent without primary antibody. Instead of commercial antibody diluent, 1x PBS with 0.1% Triton X-100 and 0.01% sodium azide can be used.</li>
	<li>Wash the kidney slice in 10 mL of 1x wash buffer overnight at RT with one change of wash buffer after 8 h.</li>
	<li>Dilute the secondary antibodies (e.g., 1:100 for Alexa Fluor-conjugated secondary antibodies) in 500 &#181;L of normal antibody diluent. Incubate the kidney slices in diluted secondary antibody for 4 days at 37 &#176;C. From this step onwards, protect the kidney slices from light.</li>
	<li>Wash the kidney slices in 10 mL of 1x wash buffer overnight at RT with one change of wash buffer after 8 h.</li>
</ol>
3. Tissue Clearing
<ol>
	<li>Transfer kidney slice to 5 mL of high grade 100% ethanol (Table of Materials) for 2 h at RT with gentle rocking (with one change to fresh ethanol after 1 h). This step is for tissue dehydration. 
	NOTE: High grade ethanol is required to achieve a high tissue translucency in the next step. Methanol or tetrahydrofuran are alternative dehydration reagents with high delipidation potential.</li>
	<li>Immerse kidney slice in 2 mL of ECi (Table of Materials) with gentle rocking at RT (with one change to fresh ECi after 2 h) overnight. 
	NOTE: The freezing/melting point of ECi is 6-8 &#176;C. Therefore, do not store samples in the fridge. Conduct immersion in a properly ventilated fume hood and avoid direct contact with clothes and skin (ECi is a non-toxic Food and Drug Administration-approved compound but has a strong odor). Use regular Eppendorf tubes or glass vessels (no polystyrene vessels).</li>
	<li>Tissue translucency can be achieved after ECi immersion and when the kidney slices are ready for imaging.</li>
</ol>
4. ConfocalImaging and Image Analysis
NOTE: For imaging, other microscopy techniques can be used as long as the refractive index matching solution is compatible with the objective lens. This protocol uses an inverted confocal microscope.
<ol>
	<li>Add 600-1,000 &#956;L of ECi into the glass bottom dish (Table of Materials). 
	NOTE: Avoid use of regular cell culture dishes, because ECi is an organic solvent that will dissolve the plastic dishes. Similarly, ECi might attack plastic parts/insulation rings on objective lenses. Refer to the appropriate reports for an overview of compatible imaging dishes30 and self-made 3-D printed chambers28.</li>
	<li>Transfer the translucent kidney slice into the dish. Place a round coverslip on the kidney slice to apply light pressure towards the glass bottom. Seal the dish with paraffin film (Table of Materials) to avoid leakage of ECi. 
	NOTE: Whole organs or several millimeter-thick tissue slices may require a border (dental cement or silicone elastomer; see Table of Materials) around the tissue to make an ECi-pool for the sample.</li>
	<li>Place the dish onto the microscope imaging platform.</li>
	<li>Take several z-stacks and perform stitching. Start with a z-step size of 5 &#956;m. 
	NOTE: Consider using long working distance (&#62;5 mm) and high numerical aperture (&#62;0.9) objectives for imaging of very thick tissue slices or organs. After imaging, transfer the tissue back to ethanol and store in wash buffer or PBS with 0.02% sodium azide.</li>
	<li>Analyze the image using 3-D rendering with software (Table of Materials).</li>
</ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Free+of+acrylamide+sodium+dodecyl+sulphate+(SDS)-based+tissue+clearing+(FASTClear):+a+novel+protocol+of+tissue+clearing+for+3-D+visualization+of+human+brain+tissues.">Free of acrylamide sodium dodecyl sulphate (SDS)-based tissue clearing (FASTClear): a novel protocol of tissue clearing for 3-D visualization of human brain tissues.</a> Neuropathology and Applied Neurobiology. 43 (4), 346-351 (2017).</li><li>Xu, N., 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=Fast+free-of-acrylamide+clearing+tissue+(FACT)-an+optimized+new+protocol+for+rapid,+high-resolution+imaging+of+3-D+brain+tissue.">Fast free-of-acrylamide clearing tissue (FACT)-an optimized new protocol for rapid, high-resolution imaging of 3-D brain tissue.</a> Scientific Reports. 7 (1), 9895 (2017).</li><li>Tainaka, 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=Whole-body+imaging+with+single-cell+resolution+by+tissue+decolorization.">Whole-body imaging with single-cell resolution by tissue decolorization.</a> Cell. 159 (4), 911-924 (2014).</li><li>Matryba, P., Bozycki, L., Pawlowska, M., Kaczmarek, L., Stefaniuk, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+perfusion-based+CUBIC+protocol+for+the+efficient+whole-body+clearing+and+imaging+of+rat+organs.">Optimized perfusion-based CUBIC protocol for the efficient whole-body clearing and imaging of rat organs.</a> Journal of Biophotonics. 11 (5), e201700248 (2018).</li></ol>

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 &amp;micro;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 G 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.
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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) ...

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

Research

JoVE Journal

Medicine

10.4K Views.  University Hospital RWTH Aachen. Groeiende interesse in het bestuderen van grote, multicellulaire structuren leidde tot de ontwikkeling van deze optische clearing techniek die het mogelijk maakt voor een driedimensionale visualisatie en kwantificering van grote structuren binnen transparante organen. Dit protocol biedt een eenvoudige, goedkope, eenvoudig op te zetten en uitgebreide tool om weefsel in drie dimensies te onderzoeken. Meerlaagse aard van een ziekte kan niet altijd worden gesimuleerd door traditionele tweedimensi...

Video: Optical Clearing and Imaging of Immunolabeled Kidney Tissue

Quantitative Visualization of Leukocyte Infiltrate in a Murine Model of Fulminant Myocarditis by Light Sheet Microscopy

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently labelled structures in large biological specimens, and is down to cellular resolution. Meanwhile, the constant refinement of underlying protocols and the enhanced availability of specialized commercial systems enable us to investigate the microstructure of whole mouse organs and even allow for the characterization of cellular behavior in various live-cell imaging approaches. Here, we describe a protocol for the spatial whole-mount visualization and quantification of the CD45+ leukocyte population in inflamed mouse hearts. The method employs a transgenic mouse strain (CD11c.DTR)that has recently been shown to serve as a robust, inducible model for the study of the development of fulminant fatal myocarditis, characterized by lethal cardiac arrhythmias. This protocol includes myocarditis induction, intravital antibody-mediated cell staining, organ preparation, and LSFM with subsequent computer-assisted image post-processing. Although presented as a highly-adapted method for our particular scientific question, the protocol represents the blueprint of an easily adjustable system that can also target completely different fluorescent structures in other organs and even in other species.

Here, we describe a light-sheet microscopy approach to visualize the cardiac CD45+ leukocyte infiltrate in a murine model of aseptic fulminant myocarditis, which is induced by the intratracheal diphtheria toxin treatment of CD11c.DTR mice.

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently ...

Immunostaining of Whole-Mount Retinas with the CLARITY Tissue Clearing Method

The tissue hydrogel delipidation method (CLARITY), originally developed by the Deisseroth laboratory, has been modified and widely used for immunostaining and imaging of thick brain samples. However, this advanced technology has not yet been used for whole-mount retinas. Although the retina is partially transparent, its thickness of approximately 200 &#181;m (in mice) still limits the penetration of antibodies into the deep tissue as well as reducing light penetration for high-resolution imaging. Here, we adapted the CLARITY method for whole-mount mouse retinas by polymerizing them with an acrylamide monomer to form a nanoporous hydrogel and then clearing them in sodium dodecyl sulfate to minimize protein loss and avoid tissue damage. CLARITY-processed retinas were immunostained with antibodies for retinal neurons, glial cells, and synaptic proteins, mounted in a refractive index matching solution, and imaged. Our data demonstrate that CLARITY&#160;can improve the quality of standard immunohistochemical staining and imaging for retinal neurons and glial cells in whole-mount preparation. For instance, 3D resolution of fine axon-like and dendritic structures of dopaminergic amacrine cells were much improved by CLARITY. Compared to non-processed whole-mount retinas, CLARITY can reveal immunostaining for synaptic proteins such as postsynaptic density protein 95. Our results show that CLARITY renders the retina more optically transparent after the removal of lipids and preserves fine structures of retinal neurons and their proteins, which can be routinely used for obtaining high-resolution imaging of retinal neurons and their subcellular structures in whole-mount preparation.

Here we present a protocol to adapt the CLARITY method of the brain tissues for whole-mount retinas to improve the quality of standard immunohistochemical staining and high-resolution imaging of retinal neurons and their subcellular structures.

The tissue hydrogel delipidation method (CLARITY), originally developed by the Deisseroth laboratory, has been modified and widely used for immunostaining ...

Neuroscience

Using 2-Photon Microscopy to Quantify the Effects of Chronic Unilateral Ureteral Obstruction on Glomerular Processes

Applying novel microscopy methods to suitable animal disease models to explore the dynamic physiology of the kidney remains a challenge. Rats with surface glomeruli provide a unique opportunity to investigate physiological and pathophysiological processes using intravital 2-photon microscopy. Quantification of glomerular capillary blood flow and vasoconstriction and dilatation in response to drugs, permeability, and inflammation are just some of the processes that can be studied. In addition, transgenic rats, i.e., podocytes labeled with fluorescent dyes and other molecular biomarker approaches, provide increased resolution to directly monitor and quantify protein-protein interactions and the effects of specific molecular alterations.
In mice, which lack surface glomeruli after four weeks of age, unilateral ureteral obstruction (UUO) for several weeks has been used to induce surface glomeruli. As this induction model does not allow for baseline studies, we quantified the effects of UUO on glomerular processes in the UUO model in Munich Wistar Fr&#246;mter (MWF) rats, which have surface glomeruli under physiologic conditions. The UUO model for five weeks or more induced significant alterations to gross renal morphology, the peritubular and glomerular microvasculature, as well as the structure and function of tubular epithelia. Glomerular and peritubular red blood cell (RBC) flow decreased significantly (p &#60; 0.01), probably due to the significant increase in the adherence of white blood cells (WBCs) within glomerular and peritubular capillaries. The glomerular sieving coefficient of albumin increased from 0.015 &#177; 0.002 in untreated MWFs to 0.045 &#177; 0.05 in 5-week-old UUO MWF rats. Twelve weeks of UUO resulted in further increases in surface glomerular density and glomerular sieving coefficient (GSC) for albumin. Fluorescent albumin filtered across the glomeruli was not reabsorbed by the proximal tubules. These data suggest that using UUO to induce surface glomeruli limits the ability to study and interpret normal glomerular processes and disease alterations.

Here, we present a protocol using 2-photon microscopy in Munich Wistar Fromter rats with surface glomeruli to quantifythe effects of prolonged ureteral obstruction on glomerular dynamics and function.

Applying novel microscopy methods to suitable animal disease models to explore the dynamic physiology of the kidney remains a challenge. Rats with surface ...

Whole-Kidney Three-Dimensional Staining with CUBIC

Although conventional pathology provided numerous information about kidney microstructure, it was difficult to know the precise structure of blood vessels, proximal tubules, collecting ducts, glomeruli, and sympathetic nerves in the kidney due to the lack of three-dimensional (3D) information. Optical clearing is a good strategy to overcome this big hurdle. Multiple cells in a whole organ can be analyzed at single-cell resolution by combining tissue clearing and 3D imaging technique. However, cell labeling methods for whole-organ imaging remain underdeveloped. In particular, whole-mount organ staining is challenging because of the difficulty in antibody penetration. The present protocol developed a whole-mount mouse kidney staining for 3D imaging with the CUBIC (Clear, Unobstructed Brain/Body Imaging Cocktails and Computational analysis) tissue clearing method. The protocol has enabled visualizing renal sympathetic denervation after ischemia-reperfusion injury and glomerulomegaly in the early stage of diabetic kidney disease from a comprehensive viewpoint. Thus, this technique can lead to new discoveries in kidney research by providing a macroscopic perspective.

The present protocol describes a tissue clearing method and whole-mount immunofluorescent staining for three-dimensional (3D) kidney imaging. This technique can offer macroscopic perspectives in kidney pathology, leading to new biological discoveries.

Although conventional pathology provided numerous information about kidney microstructure, it was difficult to know the precise structure of blood ...

ACT-PRESTO: Biological Tissue Clearing and Immunolabeling Methods for Volume Imaging

The identification and exploration of the detailed organization of organs or of the whole body at the cellular level are fundamental challenges in biology. Transitional methods require a substantial amount of time and effort to obtain a 3D image and including sectioning the intact tissue, immunolabeling, and imaging serially-sectioned tissue, which produces a loss of information at each step of the process. In recently developed approaches for high-resolution imaging within intact tissue, molecular characterization has been restricted to the labeling of proteins. However, currently available protocols for organ clearing require a considerably long process time, making it difficult to implement tissue clearing techniques in the lab. We recently established a rapid and highly-reproducible protocol termed ACT-PRESTO (active clarity technique&#8211;pressure related efficient and stable transfer of macromolecules into organs), which allows for tissue clearance within several hours. Moreover, ACT-PRESTO enables rapid immunolabeling with conventional methods and accelerates antibody penetration into the deep layer of densely-formed, thick specimens by applying pressure or convection flow. We describe how to prepare tissues, how to clear by lipid removal using electrophoresis, and how to immuno-stain by a pressure-assisted delivery. The rapidity and consistency of the protocol will expedite the performance of 3D histological research and volume-based diagnoses.

ACT-PRESTO (active clarity technique&#8211;pressure related efficient and stable transfer of macromolecules into organs) enables rapid, efficient, but inexpensive tissue clearing and rapid antibody penetration into cleared tissues by passive diffusion or pressure-assisted delivery. Using this method, a wide range of tissues can be cleared, immunolabeled by multiple antibodies, and volume-imaged.

The identification and exploration of the detailed organization of organs or of the whole body at the cellular level are fundamental challenges in ...

Biology

Application of Laser Microdissection to Uncover Regional Transcriptomics in Human Kidney Tissue

Gene expression analysis of human kidney tissue is an important tool to understand homeostasis and disease pathophysiology. Increasing the resolution and depth of this technology and extending it to the level of cells within the tissue is needed. Although the use of single nuclear and single cell RNA sequencing has become widespread, the expression signatures of cells obtained from tissue dissociation do not maintain spatial context. Laser microdissection (LMD) based on specific fluorescent markers would allow the isolation of specific structures and cell groups of interest with known localization, thereby enabling the acquisition of spatially-anchored transcriptomic signatures in kidney tissue. We have optimized an LMD methodology, guided by a rapid fluorescence-based stain, to isolate five distinct compartments within the human kidney and conduct subsequent RNA sequencing from valuable human kidney tissue specimens. We also present quality control parameters to enable the assessment of adequacy of the collected specimens. The workflow outlined in this manuscript shows the feasibility of this approach to isolate sub-segmental transcriptomic signatures with high confidence. The methodological approach presented here may also be applied to other tissue types with substitution of relevant antibody markers.

We describe a protocol for laser microdissection of sub-segments of the human kidney, including the glomerulus, proximal tubule, thick ascending limb, collecting duct and interstitium. The RNA is then isolated from the obtained compartments and RNA sequencing is carried out to determine changes in the transcriptomic signature within each sub-segment.

Gene expression analysis of human kidney tissue is an important tool to understand homeostasis and disease pathophysiology. Increasing the resolution and ...

Isolating and Imaging Live, Intact Pacemaker Regions of Mouse Renal Pelvis by Vibratome Sectioning

The renal pelvis (RP) is a funnel-shaped, smooth muscle structure that facilitates normal urine transport from the kidney to the ureter by regular, propulsive contractions. Regular RP contractions rely on pacemaker activity, which originates from the most proximal region of the RP at the pelvis-kidney junction (PKJ). Due to the difficulty in accessing and preserving intact preparations of the PKJ, most investigations on RP pacemaking have focused on single-cell electrophysiology and Ca2+ imaging experiments. Although important revelations on RP pacemaking have emerged from such work, these experiments have several intrinsic limitations, including the inability to accurately determine cellular identity in mixed suspensions and the lack of in situ imaging of RP pacemaker activity. These factors have resulted in a limited understanding of the mechanisms that underlie normal rhythmic RP contractions. In this paper, a protocol is described to prepare intact segments of mouse PKJ using a vibratome sectioning technique. By combining this approach with mice expressing cell-specific reporters and genetically encoded Ca2+ indicators, investigators may be able to more accurately study the specific cell types and mechanisms responsible for peristaltic RP contractions that are vital for normal urine transport.

The goal of this protocol is to isolate intact pacemaker regions of the mouse renal pelvis using vibratome sectioning. These sections can then be used for in situ Ca2+ imaging to elucidate Ca2+ transient properties of pacemaker cells and other interstitial cells in vibratome slices.

The renal pelvis (RP) is a funnel-shaped, smooth muscle structure that facilitates normal urine transport from the kidney to the ureter by regular, ...

3D Visualization of Immune Cell Populations in HIV-Infected Tissues via Clearing, Immunostaining, Confocal, and Light Sheet Fluorescence Microscopy

Human Immunodeficiency Virus (HIV), the causative agent of Acquired Immune Deficiency Syndrome (AIDS), is a major global health concern with nearly 40 million individuals infected worldwide and no widely accessible cure. Despite intensive efforts, a detailed understanding of virus and host cell interactions in tissues during infection and in response to therapy remains incomplete. To address these limitations, water-based tissue clearing techniques CUBIC (Clear, Unobstructed Brain/Body Imaging Cocktails and Computational analysis) and CLARITY (Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging/Immunostaining/in situ-hybridization-compatible Tissue hYdrogel) are applied to visualize complex virus host-cell interactions in HIV-infected tissues from animal models and humans using confocal and light sheet fluorescence microscopy. Optical sectioning of intact tissues and image analysis allows rapid reconstruction of spatial information contained within whole tissues and quantification of immune cell populations during infection. These methods are applicable to most tissue sources and diverse biological questions, including infectious disease and cancer.

Tissue clearing, combined with immunofluorescence microscopy, allows spatial visualization and quantification of immune-cell populations and virus proteins within intact tissues. Optical sectioning of cleared tissues with confocal and light sheet fluorescence microscopy can generate 3D models of complex tissue environments and reveal spatial heterogeneity exhibited during HIV infection.

Human Immunodeficiency Virus (HIV), the causative agent of Acquired Immune Deficiency Syndrome (AIDS), is a major global health concern with nearly 40 ...

Identification of the Source of Secreted Proteins in the Kidney by Brefeldin A Injection

Chronic kidney disease (CKD) is one of the top ten leading causes of death in the USA. Acute kidney injury (AKI), while often recoverable, predisposes patients to CKD later in life. Kidney epithelial cells have been identified as key signaling nodes in both AKI and CKD, whereby the cells can determine the course of the disease through the secretion of cytokines and other proteins. In CKD especially, several lines of evidence have demonstrated that maladaptively repaired tubular cells drive disease progression through the secretion of transforming growth factor-beta (TGF-&#946;), connective tissue growth factor (CTGF), and other profibrotic cytokines. However, identifying the source and the relative number of secreted proteins from different cell types in vivo remains challenging.
This paper describes a technique using brefeldin A (BFA) to prevent the secretion of cytokines, enabling the staining of cytokines in kidney tissue using standard immunofluorescent techniques. BFA inhibits endoplasmic reticulum (ER)-to-Golgi apparatus transport, which is necessary for the secretion of cytokines and other proteins. Injection of BFA 6 h before sacrifice leads to a build-up of TGF-&#946;, PDGF, and CTGF inside the proximal tubule cells (PTCs) in a mouse cisplatin model of AKI and TGF-&#946; in&#160;a mouse aristolochic acid (AA) model of CKD. Analysis revealed that BFA + cisplatin or BFA + AA increased TGF-&#946;-positive signal significantly compared to BFA + saline, cisplatin, or AA alone. These data suggest that BFA can be used to identify the cell type producing specific cytokines and quantify the relative amounts and/or different types of cytokines produced.

Identifying the cell type responsible for secreting cytokines is necessary to understand the pathobiology of kidney disease. Here, we describe a method to quantitatively stain kidney tissue for cytokines produced by kidney epithelial or interstitial cells using brefeldin A, a secretion inhibitor, and cell-type-specific markers.

Chronic kidney disease (CKD) is one of the top ten leading causes of death in the USA. Acute kidney injury (AKI), while often recoverable, predisposes ...

Fluorescent Labeling of Glomeruli in Cleared Mouse Kidneys for Microscopy

An Efficient and Fast Method for Labeling and Analyzing Mouse Glomeruli

The glomeruli are fundamental units in the kidney; hence, studying the glomeruli is pivotal for understanding renal function and pathology. Biological imaging provides intuitive information; thus, it is of great significance to label and observe the glomeruli. However, the glomeruli observation methods currently in use require complicated operations, and the results may lose label details or three-dimensional (3D) information. The clear, unobstructed brain imaging cocktails and computational analysis (CUBIC) tissue clearing technology has been widely used in renal research, allowing for more accurate detection and deeper detection depth. We found that mouse glomeruli can be rapidly and effectively labeled by tail vein injection of medium molecular weight FITC-Dextran followed by the CUBIC clearing method. The cleared mouse kidney could be scanned by a light-sheet microscope (or a confocal microscope when sliced) to obtain three-dimensional image stacks of all the glomeruli in the entire kidney. Processed with appropriate software, the glomeruli signals could be easily digitized and further analyzed to measure the number, volume, and frequency of the glomeruli.

Author Spotlight: Aiding Research in Kidney Biology by Labeling Glomeruli in Cleared Tissues 

This study presents an easy-to-use, complete, and simple set of methods to label and analyze glomeruli from CUBIC-cleared mouse kidneys. Data such as glomerulus number and volume can be obtained easily and reliably using fluorescein isothiocyanate (FITC)-Dextran, light sheet fluorescence microscopy (LSFM), or common confocal microscopy and software such as Imaris.

The glomeruli are fundamental units in the kidney; hence, studying the glomeruli is pivotal for understanding renal function and pathology. Biological ...

Optical Clearing and Imaging of Immunolabeled Kidney Tissue

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