Exploring Adipose Tissue Structure by Methylsalicylate Clearing and 3D Imaging

These protocol allows analysis of three-dimensional structure of Very large human on mouse adipose tissue ballet microscopy. Facilitating the linkage of pathological dysfunction with adipose tissue structural changes. This clearing process are very simple, very well adapted to clearing human on warm mice adipose tissue and use less toxic solvent such as ethanol or methyl salicylate compared to other clear protocols.

Begin by immersing the harvested mouse or human white adipose tissue in at least 10 milliliters of 4%power formaldehyde in PBS in a 15 milliliter plastic tube preferentially under a chemical hood. Shake the tissue on a rolling plate at room temperature for one hour before transferring the tube to a rolling plate at four degrees Celsius overnight. The next morning rinse the fixed white adipose tissue in 10 milliliters of PBS for five minutes at room temperature to remove all traces of fixative and transfer the tissue to a 15 milliliter tube containing 10 milliliters of 0.3%glycine in PBS for a one hour incubation at room temperature on an orbital shaker at 100 revolutions per minute.

When the remaining free aldehyde groups have been quenched, immerse the tissue in a 15 milliliter tube containing 10 milliliters of 0.2%Triton X-100 in PBS for a two hour incubation with shaking at 37 degrees Celsius. At the end of the incubation treat the tissue with 10 milliliters of 0.2%Triton X-100 and 20%DMSO with shaking at 37 degrees Celsius overnight. The next morning immerse the tissue in a 15 milliliter plastic tube containing 10 milliliters of 0.1%Tween 20, 0.1%Triton X-100, 0.1%deoxycholate and 20%DMSO in PBS for an at least 24 hour incubation at 37 degrees Celsius with shaking.

At the end of the incubation, rinse the tissue with 10 milliliters of 0.2%Triton X-100 in PBS on an orbital shaker at 100 revolutions per minute for one hour. Followed by immersion in 15 milliliters of 0.2%Triton X-100, 10%DMSO and 3%BSA in PBS for 12 hours at 37 degrees Celsius with shaking. For staining of the white adipose tissue.

Transfer the tissue into 300 microliters of 0.2%Triton X-100, 10%DMSO and 3%BSA in PBS, supplemented with the primary antibodies of interest in a 1.5 milliliter micro tube protect it from light with aluminum foil and place the tube into a 50 milliliter Falcon tube. Place the tube into the orbital shaker for a two day incubation at 37 degrees Celsius with shaking. At the end of the incubation rinse the tissue two times in 10 milliliters of 0.2%Triton X-100, 10%DMSO and 3%BSA in PBS for five hours at 37 degrees Celsius with shaking and protection from light.

After the second rinse wash the tissue in 10 milliliters of fresh 0.2%Triton X-100, 10%DMSO and 3%BSA and PBS for 16 to 48 hours at 37 degrees Celsius with shaking, then transfer the tissue to a 1.5 milliliter tube containing 300 microliters of 0.2%Triton X-100, 10%DMSO and 3%BSA in PBS, supplemented with the appropriate secondary antibodies and protect the tube from light using aluminum foil. After a two day incubation at 37 degrees Celsius on a shaker, wash the tissue two times in 10 milliliters of 0.2%Triton X-100, 10%DMSO and 3%BSA in PBS for five hours at 37 degrees Celsius with shaking protected from light. After the second wash, rinse the tissue into a tube containing 10 milliliters of fresh 0.2%Triton X-100, 10%DMSO and 3%BSA in PBS for 16 to 48 hours at 37 degrees Celsius with shaking and light protection.

Followed by a two hour wash in 10 milliliters of PBS alone at 37 degrees Celsius with shaking and light protection. For white adipose tissue clearing at the end of the PBS wash immerse the tissue and 10 milliliters of an ascending sending ethanol concentration series for a two hour incubation per concentration at room temperature with shaking protected from light as indicated. The next morning immerse the tissue in a 20 milliliter glass bottle with a plastic cap containing five milliliters of methyl salicylate in a fume hood.

The white adipose tissue is still clearly visible at this stage. Shake the container protect it from light at room temperature for at least two hours. The white adipose tissue becomes completely transplant.

For 3D confocal imaging of the stained and cleared white adipose tissue mount a metallic imaging chamber equipped with a glass bottom and in a fume hood transfer the tissue to the chamber. Fill the chamber with fresh methyl salicylate. Finalize the mountaging of the chamber by adding a small cover slip to stabilize the tissue and a large cover slip to close the chamber.

Place the chamber onto an inverted confocal microscope stage for tissue imaging at low magnification to generate a few cubic centimeter 3D maps of the whole adipose tissue sample. After 3D map generation, use a 20X long distance air objective that provides a good ratio between the resolution and depth to select several areas for tissue sampling at a higher magnification. For cell segmentation convert 3D stacks onto the software format to free up memory space.

Before opening the cell module of the software. Change the cell detection setting to plasma membrane staining and select the fluorescent channel of the marker used to delineate the cell periphery. Then set up the thresholds, provide a range of the expected cell sizes and run the segmentation.

The affects of clearing on human and mouse white adipose tissue can be observed with the naked eye. Clearing also drastically improves the image depth of the tissue that is able to be acquired. And facilitates whole tissue 3D imaging and 3D map generation out of 4X Magnification.

3D mapping further enables the selection of different areas of interest to be acquired at a 20X magnification to a depth of two millimeters. Specific staining of lipid droplets and adipocytes can be achieved using perilipin and anti-GLUT4 antibodies respectively. Blood vessels can be detected using either CD31 antibody or intravenous injection of Lectin DyLight 649 shortly before sacrifice.

Macrophages and T-cells can be visualized using anti CD301 phycoerythrin antibody and anti TCR beta Pacific blue antibody. The peripheral nerve network can be detected using anti Tyrosine Hydroxylase antibody. Using these labelings the mean size and size distribution of the adipocytes and the blood vessel network density within the adipose tissue can then be determined.

It's crucial to follow occupation time as demonstrated because poor sample preparation will not result in a good image quality. This protocol can be combined with advanced imaging system or can be coupled to post-processing image analysis freeware.