tissue clearing of mouse kidneys for confocal microscopy

Fluoreszenzmarkierung von Glomeruli in geklärten Mausnieren für die Mikroskopie

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.

Autor im Rampenlicht: Unterstützung der Forschung in der Nierenbiologie durch Markierung von Glomeruli in geklärten Geweben 

Eine effiziente und schnelle Methode zur Markierung und Analyse von Mausglomeruli

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  

Gewebereinigung von Mäusenieren für die konfokale Mikroskopie

Zu Beginn werden die präparierten und fixierten Mäusenieren in PBS gelegt. Schneiden Sie die Niere mit Hilfe der Gehirnmatrix in einen Millimeter dicke Abschnitte. Die Scheiben in 4%Paraformaldehyd überführen und über Nacht bei vier Grad Celsius fixieren.Für die Lichtblattmikroskopie fixieren Sie die gesamte Niere, indem Sie sie in 4%PFA eintauchen und 48 Stunden lang bei vier Grad Celsius inkubieren. Für die Gewebereinigung sammeln Sie die Nierenproben in einem 50-Milliliter-Zentrifugenröhrchen und reinigen sie dann in Kubik-L mit Schütteln bei 60 U/min bei 37 Grad Celsius. Tauschen Sie kubisches L intermittierend aus, bis eine zufriedenstellende optische Transparenz erreicht ist.Waschen Sie die Proben dreimal in PBS unter leichtem Schütteln bei Raumtemperatur für jeweils eine Stunde. Tragen Sie dann kubisches R auf die Proben auf und legen Sie sie sechs Stunden lang zum Schütteln hin. Beobachten Sie die gereinigten Proben direkt oder lagern Sie sie eine Woche lang in undurchsichtigen, mit kubischem R gefüllten Röhrchen bei Raumtemperatur.

tissue-clearing-of-mouse-kidneys-for-confocal-microscopy

Research

JoVE Journal

Biology

Tissue Clearing of Mouse Kidneys for Confocal Microscopy

277 Aufrufe. Zu Beginn werden die präparierten und fixierten Mäusenieren in PBS gelegt. Schneiden Sie die Niere mit Hilfe der Gehirnmatrix in einen Millimeter dicke Abschnitte. Die Scheiben in 4%Paraformaldehyd überführen und über Nacht bei vier Grad Celsius fixieren.Für die Lichtblattmikroskopie fixieren Sie die gesamte Niere, indem Sie sie in 4%PFA eintauchen und 48 Stunden lang bei vier Grad Celsius inkubieren. Für die Gewebereinigung sammeln Sie die Nierenproben in einem 50-Milliliter-Ze...

Serie: 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 ...