Labeling and counting glomeruli using conventional methods can be complicated, tedious, time consuming, and bulky instruments 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. To begin, place the dissected and fixed mouse kidneys in PBS.
Slice the kidney into one-millimeter thick sections using the brain matrix. Transfer the slices to 4%paraformaldehyde and fix at four-degrees Celsius overnight. For light sheet microscopy, fix the whole kidney by submerging it in 4%PFA and incubating it at four-degrees Celsius for 48 hours.
For tissue clearing, collect the kidney samples in a 50-milliliter centrifuge tube and then clear them in CUBIC-L, with shaking at 60 RPM at 37-degrees Celsius. Replace CUBIC-L intermittently until satisfactory optical transparency is achieved. Wash the samples three times in PBS, with gentle shaking at room temperature for one hour each.
Then, apply CUBIC-R to the samples and place them for shaking for six hours. Observe the cleared samples directly, or store them in opaque tubes filled with CUBIC-R at room temperature for one week. To begin, prepare clear kidney tissue samples.
Then, turn on the confocal microscope by first activating the laser power and then engaging the confocal control switch. Next, start the computer, power up the microscope, and initiate the confocal hardware. After running the self test, launch the confocal software.
Select the appropriate lens. Retrieve the sample from the CUBIC-R reagent and place it in the confocal dish. Cover it to prevent the CUBIC-R reagent from drying.
Move the sample to the center of the field of view and adjust the focus until the sample is in focus. For 3D reconstruction, adjust the zoom plane in one direction until no fluorescence is visible, defining this position as the top. Then, adjust the zoom plane in the opposite direction until no fluorescence is visible, defining this position as the bottom.
Click the Run button in the lower-right corner of the dialogue box to initiate the scanning. For large image reconstruction, position the field of view in the middle of the sample and establish this as the center. Choose a two-by-two field of view.
In the ND multifunction interface, select the Z-stack to reconstruct large images. Adjust the various options in the panel as needed, and press the Run button to begin the scanning. Adhere the transparent kidney to the sample-fixing adapter with glue and wait until the kidney is securely attached.
Then, immerse the kidney in the sample bin, and slide the sample bin into the microscope system. Seal the hatch. In the Locate interface, adjust the sample to an appropriate observation position.
Switch to the Acquisition interface. Set the optical path. Select the 488-nanometer laser.
Choose light beam filter of 405, 488, 561, 640. Then, set the optical splitting filter block to SBS LP 490. Select camera two and alter the pseudo-color to green.
Input the left and then right Z-offset obtained from the channel submenu. Select the smallest zoom and fine tune it for optical clarity. Identify the XY-boundary and Z-range for the entire organ.
Then, modulate the laser intensity and exposure time to less than 10 milliseconds. Initiate the process by clicking Start Experiment. If the tissue is excessively large, combine two or more images with image stitching using the microscopy software.
Open the captured file and select Processing, then Method, and Stitching, followed by Method Parameter. Click Apply to finalize image stitching. Intravascularly-injected FITC-dextran was utilized for labeling the glomeruli.
In the cleared kidney, the glomeruli could be clearly observed by using light sheet microscopy or confocal microscopy. To begin, capture images of labeled glomeruli under a confocal or light sheet fluorescence microscope. Import the image files into the image-analysis software.
Click the Surface button to create a new surface. Follow the guide at the bottom-left part of the screen. Click the Next button.
Tick the box for the Smooth option, and enter a number 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.
Adjust if needed and click the Next button. Select the proper range and click the Next button to create a surface. Then, click Filter, followed by Add button to add Sphericity to filter out non-spherical objects.
Click the Duplicate button to copy this new surface. Click the Statistics button, followed by the Overall button. The software will present the number of glomeruli as the total number of surface.
To calculate the volume of glomeruli, click the Selection button to select the target objects and present the volume of every object. Export statistics to a spreadsheet for further analysis. To calculate the volume of the slice or whole kidney, first, import the images into the image-analysis software.
After clicking the Surface button and entering a number in the Smooth option, select Absolute Intensity in Thresholding. Click the Next button. Adjust the surface until it covers the whole tissue, and click the Next button to create the surface.
Click the Selection button to select the surface of the whole tissue and present the volume of the tissue. Finally, export the statistics to a spreadsheet. Using image-processing software, the number and volume of glomeruli was counted in a kidney slice, in an entire kidney, or in a strip.
Using these values, the volume ratio and frequency of the glomeruli could be calculated.
