Optical Clearing and Imaging of Immunolabeled Kidney Tissue

Summary

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.

Abstract

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.

Introduction

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 dimensions^1,2,3. 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 imaging^5,6,7.

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 immersion^8,9, hyperhydration^10,11, and hydrogel embedding^12,13. 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 dehydration^{14,15,16,17,18,19,20,21}. 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 1.38-1.52^{8,11,12,22,23,24}. 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 results^7,26.

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 glomeruli^27,28, 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.

Protocol

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

1. Prepare solutions the same day or evening before and store in a fridge overnight. Warm solutions to room temperature (RT) before using.
2. Make a fresh batch of 3% paraformaldehyde (PFA) in 1x phosphate-buffered saline (PBS). About 50-100 mL PFA is needed per mouse.
   1. 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 °C. Do not heat above 70 °C.
      CAUTION: PFA is toxic. Prepare PFA in the fume hood.
   2. 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.
   3. Remove the solution from heat. Add 50 mL of 20x PBS. Chill on ice to RT.
   4. Adjust the pH to 7.3-7.4 with HCl. Add distilled water to 1 L.
   5. Remove any undissolved particles by filtering 1x PBS and 3% PFA with 0.22 µm filter.
3. 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.
4. 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.
5. Label a 15 mL conical tube and dispense 10 mL of PFA into it.
6. 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).
7. 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.
8. 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.
9. 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.
10. 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.
11. Collect the perfused kidney carefully and avoid puncturing or squeezing the tissue.
12. 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.
13. Immerse the kidney slices with the prepared PFA in the labeled 15 mL conical tube.
14. 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.
15. Carry out post-fixation overnight at RT protected from light.

2. Tissue Preparation and Immunostaining

1. After post-fixation, wash the kidney slice twice with 1x wash buffer (Table of Materials) for 1 h on a horizontal rocker at RT.
2. Perform antigen retrieval. Heat up 300 mL of 1x antigen unmasking solution (Table of Materials) in a 500 mL beaker to 92-98 °C. Enclose the slice in embedding cassette permeable to the heated buffer with stirring for 1 h at 92-98 °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).
3. 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.
4. Dilute the primary antibody in 500 µL of normal antibody diluent (Table of Materials). Begin with a concentration of ­­­1:50-1:100. Gently rock the kidney slice in diluted primary antibody for 4 d at 37 °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.
5. Wash the kidney slice in 10 mL of 1x wash buffer overnight at RT with one change of wash buffer after 8 h.
6. Dilute the secondary antibodies (e.g., 1:100 for Alexa Fluor-conjugated secondary antibodies) in 500 µL of normal antibody diluent. Incubate the kidney slices in diluted secondary antibody for 4 days at 37 °C. From this step onwards, protect the kidney slices from light.
7. Wash the kidney slices in 10 mL of 1x wash buffer overnight at RT with one change of wash buffer after 8 h.

3. Tissue Clearing

1. 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.
2. 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 °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).
3. Tissue translucency can be achieved after ECi immersion and when the kidney slices are ready for imaging.

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.

1. Add 600-1,000 μL 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.
2. 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.
3. Place the dish onto the microscope imaging platform.
4. Take several z-stacks and perform stitching. Start with a z-step size of 5 μm.
   NOTE: Consider using long working distance (>5 mm) and high numerical aperture (>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.
5. Analyze the image using 3-D rendering with software (Table of Materials).

Results

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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Discussion

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...

Materials

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