tissue clearing of mouse kidneys for confocal microscopy

Mikroskopi için Temizlenmiş Fare Böbreklerinde Glomerüllerin Floresan Etiketlenmesi

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.

Yazar Spotlight: Temizlenmiş Dokularda Glomerülleri Etiketleyerek Böbrek Biyolojisi Araştırmalarına Yardımcı Olmak 

Fare Glomerüllerini Etiketlemek ve Analiz Etmek için Etkili ve Hızlı Bir Yöntem

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  

Konfokal mikroskopi için fare böbreklerinin doku temizliği

Başlamak için, disseke ve sabit fare böbreklerini PBS'ye yerleştirin. Beyin matrisini kullanarak böbreği bir milimetre kalınlığında bölümlere ayırın. Dilimleri %4 paraformaldehite aktarın ve gece boyunca dört santigrat derecede sabitleyin.Işık tabakası mikroskobu için, tüm böbreği %4 PFA'ya batırarak ve 48 saat boyunca dört santigrat derecede inkübe ederek sabitleyin. Doku temizliği için, böbrek örneklerini 50 mililitrelik bir santrifüj tüpünde toplayın ve daha sonra 37 santigrat derecede 60 RPM'de çalkalayarak kübik L cinsinden temizleyin. Tatmin edici optik şeffaflık elde edilene kadar kübik L'yi aralıklı olarak değiştirin.Numuneleri PBS'de her biri bir saat oda sıcaklığında hafifçe çalkalayarak üç kez yıkayın. Daha sonra numunelere kübik R uygulayın ve altı saat çalkalamaya bırakın. Temizlenmiş numuneleri doğrudan gözlemleyin veya bir hafta boyunca oda sıcaklığında kübik R ile doldurulmuş opak tüplerde saklayın.

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

Research

JoVE Journal

Biology

Tissue Clearing of Mouse Kidneys for Confocal Microscopy

277 Görüntüleme. Başlamak için, disseke ve sabit fare böbreklerini PBS'ye yerleştirin. Beyin matrisini kullanarak böbreği bir milimetre kalınlığında bölümlere ayırın. Dilimleri %4 paraformaldehite aktarın ve gece boyunca dört santigrat derecede sabitleyin.Işık tabakası mikroskobu için, tüm böbreği %4 PFA'ya batırarak ve 48 saat boyunca dört santigrat derecede inkübe ederek sabitleyin. Doku temizliği için, böbrek örneklerini 50 mililitrelik bir santrifüj tüpünde toplayı...

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 ...