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 ....

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 ....

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

List of materials used in this article

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)