This study was supported by grants from the National Natural Science Foundation of China (82204951) and Sichuan Science and Technology Program (2020JDRC0102).

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
<ol>
	<li>Glomeruli labeling
	<ol>
		<li>Dissolve FITC-dextran (10 mg) in 1x phosphate-buffered saline (PBS) at a ratio of 1:1 (1 mg: 1 mL) to p...

Fluorescent Labeling of Glomeruli in Cleared Mouse Kidneys for Microscopy

<ol>
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G., 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=Glomerular+number+and+size+variability+and+risk+for+kidney+disease.">Glomerular number and size variability and risk for kidney disease.</a> Current Opinion in Nephrology and Hypertension. 20 (1), 7-15 (2011).</li><li>Bertram, J. F., 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=Why+and+how+we+determine+nephron+number.">Why and how we determine nephron number.</a> Pediatric Nephrology. 29, 575-580 (2014).</li><li>Bertram, J. F., Soosaipillai, M. C., Ricardo, S. D., Ryan, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Total+numbers+of+glomeruli+and+individual+glomerular+cell+types+in+the+normal+rat+kidney.">Total numbers of glomeruli and individual glomerular cell types in the normal rat kidney.</a> Cell and Tissue Research. 270 (1), 37-45 (1992).</li><li>Nyengaard, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stereologic+methods+and+their+application+in+kidney+research.">Stereologic methods and their application in kidney research.</a> Journal of the American Society of Nephrology. 10 (5), 1100-1123 (1999).</li><li>Bertram, J. 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All authors declare no conflict of interest.

Tissue-clearing technologies can be classified into 3 or 4 groups29,30,31. 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...

The number and volume of glomeruli are very important for the diagnosis and treatment of various kidney diseases1,2,3,4,5. 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 expensive6,7,8,9. Biopsy provides a wealth of information, but obviously, this method is only suitable for rough estimations10,11. Medical imaging technologies, including magnetic resonance imaging (MRI), computed tomography (CT), and X-ray, are also widely used in glomerular detection12,13,14,15, 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 organs18,19,20,21,22,23. 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>4% PFA</td>
			<td>Biosharp</td>
			<td>7007171800</td>
			<td>Fixation reaagen</td>
		</tr>
		<tr>
			<td>502 Glue&#160;</td>
			<td>Deli</td>
			<td>7146</td>
			<td>For fixing the kidney to the sample fixing adapter&#160;</td>
		</tr>
		<tr>
			<td>Antipyrine</td>
			<td>Aladdin</td>
			<td>A110660</td>
			<td>Clearing reagent</td>
		</tr>
		<tr>
			<td>Brain Matrix</td>
			<td>RWD Life Science</td>
			<td>1mm 40-75</td>
			<td>Tissue slicing</td>
		</tr>
		<tr>
			<td>Confocal microscopy</td>
			<td>Nikon</td>
			<td>A1plus</td>
			<td>Image acquisition</td>
		</tr>
		<tr>
			<td>FITC-Dextran</td>
			<td>Sigma-Aldrich</td>
			<td>FD150S</td>
			<td>Labeling reagent</td>
		</tr>
		<tr>
			<td>Light sheet fluorescence microscopy&#160;</td>
			<td>Zeiss</td>
			<td>Light sheet 7&#160;</td>
			<td>Image acquisition</td>
		</tr>
		<tr>
			<td>Mice</td>
			<td>Ensiweier</td>
			<td></td>
			<td>Adult C57BL/6 mice (6 weeks of age, 25&#8211;30 g)&#160;</td>
		</tr>
		<tr>
			<td>N-Butyldiethanolamine</td>
			<td>Aladdin</td>
			<td>B299095</td>
			<td>Clearing reagent</td>
		</tr>
		<tr>
			<td>Nicotinamide</td>
			<td>Aladdin</td>
			<td>N105042</td>
			<td>Clearing reagent</td>
		</tr>
		<tr>
			<td>Pentobarbital Natriumsalz</td>
			<td>Sigma-Aldrich</td>
			<td>P3761</td>
			<td></td>
		</tr>
		<tr>
			<td>Tail vein fixator</td>
			<td>JINUOTAI</td>
			<td>JNT-FS35</td>
			<td>Fix the mouse for vail injection</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T8787</td>
			<td>Clearing reagent</td>
		</tr>
	</tbody>
</table>

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 

An Efficient and Fast Method for Labeling and Analyzing Mouse Glomeruli

Labeling and counting glomeruli using conventional methods can be complicated, tedious, time consuming, and bulky instrument are required. Our method offers an instant and intuitively accurate labeling method for analysis of the glomeruli within the entire kidney. Our technique is a quick, easy, accurate, and cheap method to analyze glomeruli in the entire kidney.It does not require any special equipment, so it can be carried out in any laboratory. This is a groundbreaking, rapid, and simple method to label glomeruli, allowing for an intuitive observation of all the glomeruli within the entire kidney. It holds great potential to assist researchers to make significant breakthroughs in the study of the kidney by providing information such as glomeruli quantity, volume, distribution, and more.On one hand, we're exploring the application of this glomeruli labeling method in clinical kidney disease research. On the other hand, we'll keep working on enhancing methods to label biotargets like blood vessels or specific proteins in transparent tissues, adjusting them to meet our research needs.

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...

Watch this Scientific Journal Video about Aiding Research in Kidney Biology by Labeling Glomeruli in Cleared Tissues at JoVE.com

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 ...

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Research

JoVE Journal

Biology

Tissue Clearing of Mouse Kidneys for Confocal Microscopy

Confocal and Light Sheet Fluorescence Microscopy (LSFM) Imaging of Cleared Mouse Kidneys

Data Processing and Quantification of Glomeruli from Microscopy Images

690 Views.  Sichuan University.  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.

Fluorescent Labeling of Glomeruli in Cleared Mouse Kidneys for Microscopy

Özet