tissue clearing of mouse kidneys for confocal microscopy

Marquage fluorescent de glomérules dans des reins de souris dégagés pour la microscopie

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.

Pleins feux sur l’auteur : Aider la recherche en biologie rénale en étiquetant les glomérules dans les tissus clairsemés 

Une méthode efficace et rapide pour l’étiquetage et l’analyse des glomérules de souris

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.

Watch this Scientific Journal Video about Tissue Clearing of Mouse Kidneys for Confocal Microscopy at JoVE.com

Tissue Clearing of Mouse Kidneys for Confocal Microscopy  

Nettoyage tissulaire des reins de souris pour la microscopie confocale

Pour commencer, placez les reins de souris disséqués et fixés dans PBS. Coupez le rein en sections d’un millimètre d’épaisseur à l’aide de la matrice cérébrale. Transférez les tranches à 4% de paraformaldéhyde et fixez-les à quatre degrés Celsius pendant la nuit.Pour la microscopie à feuille optique, fixez le rein entier en l’immergeant dans 4 % de PFA et en l’incubant à quatre degrés Celsius pendant 48 heures. Pour le nettoyage des tissus, prélever les échantillons de rein dans un tube à centrifuger de 50 millilitres, puis les nettoyer dans un L cubique en agitant à 60 tr/min à 37 degrés Celsius. Remplacez le L cubique par intermittence jusqu’à ce que la transparence optique soit satisfaisante.Lavez les échantillons trois fois dans du PBS en agitant doucement à température ambiante pendant une heure chacun. Appliquez ensuite R cubique aux échantillons et placez-les pour les agiter pendant six heures. Observez directement les échantillons dégagés ou stockez-les dans des tubes opaques remplis de R cubique à température ambiante pendant une semaine.

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Research

JoVE Journal

Biology

Tissue Clearing of Mouse Kidneys for Confocal Microscopy

272 Views.  Sichuan University. Tissue Clearing of Mouse Kidneys for Confocal Microscopy

Video: Tissue Clearing of Mouse Kidneys for Confocal 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.

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 ...