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

1. Glomeruli labeling
   1. Dissolve FITC-dextran (10 mg) in 1x phosphate-buffered saline (PBS) at a ratio of 1:1 (1 mg: 1 mL) to prepare the working probe solution.
      NOTE: The working solution can be stored at 4 °C for 1 month.
   2. Place the C57 mouse in a mouse tail vein fixator. Wet the gauze with hot water, wrap the gauze around the middle part of the mouse tail, and warm the tail for 1 min.
   3. Wipe the mouse tail with 75% ethanol until the left and right tail veins are visible.
   4. Draw 100 µL of the working probe solution with a 1 mL insulin syringe and slowly inject the solution into the mouse tail vein.
   5. After injection, let the probe solution circulate for 30 min. Allow the mice to move freely in their cages.
   6. Once sufficient circulation is achieved, deeply anesthetize the miceby intraperitoneal injection of sodium pentobarbital (1%, 60-80 µg/kg).
2. Kidney sampling and fixation
   1. Fix the anesthetized mice to the anatomical plate in a supine position. Secure their paws with medical tape.
   2. After skin preparation, euthanize the mice with a lethal dose of sodium pentobarbital (1%, 10 mg/kg).
   3. Make a midline abdominal incision to expose the abdominal cavity.
   4. Reveal the kidneys and remove them with a scalpel and scissors in the fume hood. Remove the renal envelope gently.
   5. Collect the kidneys and fix them in 4% paraformaldehyde (PFA) at 4 °C overnight.
   6. Wash the samples with PBS three times 1 h each with shaking at room temperature (RT).
   7. Prepare the kidney for microscopy.
      1. Slice the kidney for confocal microscopy: Cut the kidney into 1 mm thick slices using the brain matrix (1 mm steel, 40-75 g) to standardize the slice thickness. Transfer the slices into 4% PFA and continue the fixation at 4 °C overnight.
      2. Whole kidney for light-sheet microscopy: Fix the kidneys in 4% PFA at 4 °C for 48 h.

2. Clearing

1. Reagent preparation
   1. Prepare CUBIC-L reagent by mixing 10% (wt/wt) N-butyldiethanolamine, 10% (wt/wt) Triton X-100 and 80% ddH₂O.
   2. Prepare CUBIC-R reagent by mixing 45% (wt/wt) antipyrine, 30% (wt/wt) nicotinamide, 0.5% (vol/vol) N-butyldiethanolamine and 24.5% ddH₂O. Ensure the solution has a pH of 9.6-9.8 and a refractive index (RI) of 1.522.
      NOTE: CUBIC-L is used for delipidation, and CUBIC-R is used for refractive index matching.
2. Clearing procedure
   1. Collect the kidney samples in a 50 mL centrifuge tube and then clear them in CUBIC-L with shaking at 60 rpm at 37 °C. Replace CUBIC-L every 4 h (for kidney slices) or every 24 h (for a whole kidney) until achieving satisfactory optical transparency.
      ​NOTE: This process takes 1 day for kidney slices and 5 days for a whole kidney.
   2. Wash the samples three times in PBS with gentle shaking at RT for 1 h each.
   3. Apply CUBIC-R to the samples, shaking samples at 37 °C, 60 rpm for 6 h.
   4. Observe the cleared samples directly or store them in black tubes filled with CUBIC-R at RT for 1 week.

3. Image acquisition

1. Confocal imaging
   NOTE: Image the sliced kidney samples with confocal microscopy (Lenses: A Plan Apo λ 4×/0.2 N1 objective or a Plan Apo VC 10×/0.45 DIC N1 objective). Capture large images (e.g., 6052 µm x 6052 µm for the kidney slice) with z-stacks.
   1. Turn on the confocal microscope in the following sequence: laser power, confocal control switch, computer, microscope, and confocal hardware. After running the self-test, turn on the confocal software.
   2. Select the appropriate lens. Remove the sample from the CUBIC-R reagent, place it in the confocal dish, and cover it to prevent the CUBIC-R reagent from drying out.
   3. Move the sample to the middle of the field of view and adjust the focal plane until it is in focus.
   4. Apply 3D reconstruction (step 3.1.4.1) and large image reconstruction (steps 3.1.4.2-3.1.4.3).
      1. 3D reconstruction (confocal): Switch the zoom plane in one direction until no fluorescence is visible; this position is defined as the top. Then, switch the zoom plane in the opposite direction until no fluorescence is visible; this position is defined as the bottom. Click the Run button in the lower-right corner of the dialog box to start the scanning.
      2. Large image reconstruction (confocal): Move the field of view to the middle of the sample, and set the current position as the center. Select a 2 x 2 field of view.
      3. In the ND multifunction interface, select the Z stack to reconstruct large images and set different options in the panel. Then press the Run butto n to start the scanning.
2. Light sheet fluorescence microscopy (LSFM) imaging
   NOTE: Image the whole kidneys with a light sheet fluorescence microscope (Lens: EC Plan-Neofluar 5x/0.16 (Medium n = 1.529) objective).
   1. Sample attaching (LSFM):
      1. Glue the transparent kidney to the sample fixing adapter and wait until the kidney is firmly attached.
      2. Soak the kidney in the sample bin and push the sample bin into the microscope system. Close the hatch.
   2. Observe and scan (LSFM):
      1. In the Locate interface, move the sample to a suitable observation position. Switch to the Acquisition interface, set the optical path, select 488 nm laser, LBF 405/488/561/640, optical splitting filter block SBS LP 490, select Camera 2, and change the pseudo-color to green.
      2. Fill in the Left/Right Z Offset value recorded earlier in the Channel submenu. Select the smallest zoom (0.36) and fine-tune it for maximum clarity.
   3. Find the X\Y boundary and Z range of the entire organ. Adjust the laser intensity and exposure time (preferably less than 10 ms), and click Run to start.
   4. If the tissue is too big, merge two or more images with image stitching. Open the captured file using the microscopy software (here, ZEISS ZEN 3.7). Select Processing > Method > Stitching > Method Parameter > Apply to complete image stitching.

4. Data processing and quantification

NOTE: Process the image stacks with Imaris (image analysis) software, using the Surface function to label the glomeruli and perform analysis.

1. Count of glomeruli
   1. Import large images (confocal microscopy) or stitched images (light-sheet microscopy) into the image analysis software.
   2. Open the files in the image analysis software. Click the Surface button to create a new Surface. Follow the guide at the bottom left part of the screen.
      1. Click the Next button (right arrow button) without ticking anything.
      2. Tick the box before the Smooth option and enter a proper number (e.g., 14.5) to control the detail of the Surface. Select Background Subtraction in Thresholding and fill the box with the approximate average diameter of the glomeruli. Click the Next button.
      3. Adjust if needed, and click the Next button. Select the proper range and click the Next button to create a Surface.
   3. Click Filter, then Add button to add Sphericity to filter out non-spherical objects. Click the Duplicate button to copy this new surface.
   4. Click the Statistics button, then click the Overall button; the software will present the number of glomeruli as the Total Number of Surface.
2. Volume of glomeruli
   1. Create a surface of the glomeruli as above.
   2. When the Surface is completed, click the Selection button to select the target objects and present the volume of every object. Click the Save button, and export statistics to a spreadsheet for further analysis.
3. Volume of the slice or whole kidney
   1. Import large images (confocal microscopy) or stitched images (light-sheet microscopy) into the image analysis software.
   2. Open files in the image analysis software. Click the Surface button to create a new Surface. Follow the guide at the bottom left part of the screen.
      1. Click the Next button (right arrow button) without ticking.
      2. Tick the box before the Smooth option and enter a proper number (e.g., 50) to control the detail of the Surface. Select Absolute Intensity in Thresholding. Click the Next button.
      3. Adjust the Surface until it covers the whole tissue, and click the Next button. Click the Next button to create the Surface.
   3. Click the Selection button to select the Surface of the whole tissue and present the volume of the tissue. Click the Save button to export statistics to a spreadsheet.

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

Name	Company	Catalog Number	Comments
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