An Efficient and Fast Method for Labeling and Analyzing Mouse Glomeruli

Summary

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.

Abstract

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.

Introduction

The number and volume of glomeruli are very important for the diagnosis and treatment of various kidney diseases^1,2,3,4,5. The golden standard of glomeruli number estimation is the physical dissector/fractionator combination. However, this method requires special reagents and equipment, making it slow and expensive^6,7,8,9. Biopsy provides a wealth of information, but obviously, this method is only suitable for rough estimations^10,11. Medical imaging technologies, including magnetic resonance imaging (MRI), computed tomography (CT), and X-ray, are also widely used in glomerular detection^12,13,14,15, but such technologies require bulky instruments. New methods, such as matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometer¹⁶ or the thick and thin section method¹⁷, have also been used in glomerular detection, though they remain tedious and laborious.

With the help of transparency technologies, it is possible to observe deeper depths and obtain richer and more complete information from thick tissues or even whole organs^{18,19,20,21,22,23}. Therefore, transparency technologies have been widely used in kidney research²⁴. The observation and detection of glomeruli in the cleared kidneys are also involved. However, these published articles either only briefly referred to glomerular detection²⁵ or used difficult-to-achieve labeling methods such as transgenic animals²⁶, self-produced dyes¹³, or high-concentration antibody incubation²⁷ to label the glomeruli. In addition, although studies had analyzed glomeruli in cleared kidneys, the analyses were always limited¹³ or relied on analysis algorithms established by the authors themselves²⁶.

We have previously demonstrated a more convenient way to label the glomeruli in mice kidneys²⁸. By using Imaris, we found that glomeruli count, frequency, and volume could be quickly obtained. Thus, here we present a more accessible, comprehensive, and simplified set of methods to label and analyze the glomeruli of mice kidneys.

Protocol

Adult C57BL/6 mice (6 weeks of age, 25-30 g) were used in this study. All procedures were performed in compliance with local regulations of animal welfare and experimental ethics. The study was approved by the West China Hospital of Sichuan University Biomedical Research Ethics Committee. 1. Glomeruli labeling and tissue preparation Glomeruli labeling Dissolve FITC-dextran (10 mg) in 1x phosphate-buffered saline (PBS) at a ratio of 1:1 (1 mg: 1 mL) to p…

Representative Results

This study provides a simple and efficient method for labeling and analyzing the glomeruli in mice kidneys. Glomeruli (blood vessels) can be well labeled by intravascularly injected FITC-Dextran. After the clearing process, the kidney became transparent (Figure 1A), and the glomeruli could be clearly observed by using light-sheet microscopy (Figure 1B) or confocal microscopy (Figure 1C). Confocal microsco…

Discussion

Tissue-clearing technologies can be classified into 3 or 4 groups^29,30,31. Organic solvent-based tissue clearing (e.g., DISCO and PEGASOS), aqueous-based tissue clearing (e.g., CUBIC), and hydrogel embedding tissue clearing (e.g., CLARITY) have all been applied in kidney clearing ^25,26,28,32. CUBIC, as…

Materials

4% PFA	Biosharp	7007171800	Fixation reaagen
502 Glue	Deli	7146	For fixing the kidney to the sample fixing adapter
Antipyrine	Aladdin	A110660	Clearing reagent
Brain Matrix	RWD Life Science	1mm 40-75	Tissue slicing
Confocal microscopy	Nikon	A1plus	Image acquisition
FITC-Dextran	Sigma-Aldrich	FD150S	Labeling reagent
Light sheet fluorescence microscopy	Zeiss	Light sheet 7	Image acquisition
Mice	Ensiweier		Adult C57BL/6 mice (6 weeks of age, 25–30 g)
N-Butyldiethanolamine	Aladdin	B299095	Clearing reagent
Nicotinamide	Aladdin	N105042	Clearing reagent
Pentobarbital Natriumsalz	Sigma-Aldrich	P3761
Tail vein fixator	JINUOTAI	JNT-FS35	Fix the mouse for vail injection
Triton X-100	Sigma-Aldrich	T8787	Clearing reagent