Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue

This method can help answer key questions in the field of Adipose Tissue Biology, such as how different components of Adipose Tissue interact in an entire tissue. The main advantage of this technique is that it fully removes the excess lipids stored in Adipose Tissue while maintaining it’s three-dimensional architecture. While this method can provide insight into those three-dimensional organization of Adipose Tissue it can also be generally applied to other tissues with high lipid content such as memory gland and lymph node.

Visual demonstration of this method is critical as the delipidation and the clearing steps require sufficient incubation times, which can be difficult to determine. Begin by using blunt ended forceps to dissect the fat pad of interest from a freshly perfused animal model. Carefully remove the entire fad pad without pinching or squeezing the tissue or contamination the dissected tissue with fur.

Place the fat pad in approximately 10 milliliters of fixation solution in a 15 milliliter conical tube and post fix at four degrees celsius overnight. The next day after washing the sample with PBS, gently remove any debris from the sample under a dissection microscope. Hair, lint, or other debris attached to the sample will cause shadows during imaging.

Transfer the clean sample to a polypropylene tube containing ice cold, 20 percent methanol B1N buffer. Place the tube containing the sample horizontally on an orbital shaker, set it 100 RPM, and shake for 30 minutes to 1 hour depending on the size of the sample. Ensure the samples can move freely inside the tube.

Continue dehydrating the samples through a graded series of 40 percent, 60 percent, and 80 percent methanol in B1N buffer followed by 100 percent methanol. Minimize the carryover solution. Following dehydration, replace the 100 percent methanol solution with 100 percent dichloromethane or DCM to delipidate the sample.

Repeat the wash twice following the incubation times listed in table 2. The sample should sink to the bottom of the tube at the end of the second and third DCM washes. If not, extend the incubation time to ensure full delipidation.

After delipidation, wash twice with 100 percent methanol for the time indicated in table 2. Rehydrate the sample in a reversed methanol B1N buffer series for the times indicated in table 2. After rehydration, wash the sample with PTXWH buffer for 2 hours at room temperature.

The sample is now ready for immunostaining or could be stored in PTXWH buffer at 4 degrees celsius for up to 1 year. First, dilute the primary antibody in PTXWH buffer to the recommended concentration. Then, centrifuge the diluted antibody solution at 20 thousand G for 10 minutes to prevent introducing antibody precipitates or aggregations.

It is important to choose proper primary antibodies that are compatible with methanol and DCM treatment. New antibodies should be validated with small pieces of tissue or with methanol treated tissue sections. incubate the delipidated sample with the primary antibody solution for 3 to 5 days depending on the size of the sample.

After the primary incubation wash the sample with several changes of PTXWH buffer. The next day, dilute the secondary antibody in PTXWH buffer to the recommended concentration. Centrifuge the diluted antibody solution as before.

Incubate the sample with the secondary antibody solution for 3 to 5 days depending on the size of the sample. Again, wash the sample with PTXWH buffer in a series of incubation steps as before. The next day, wash the sample with 1XPBS for 5 minutes, 10 minutes, then 30 minutes.

Embed Adipose Tissue in agarose to facilitate sample mounting for light sheet microscopy. First, prepare the embedding solution with 1 percent agarose in 1XPBS. Cool the embedding solution to around 40 degrees celsius to avoid exposing the sample to excessive heat.

While avoiding liquid carryover, place the cleaned tissue sample into a mold and arrange it into the desired position. Then gently pour the embedding solution over the sample while avoiding air bubbles. Any air bubbles should be moved to the periphery before the agarose solidifies.

Once the agarose has fully solidified, cut out a block containing the sample. Transfer the sample to 25 percent methanol in water, protect from light, and incubate with shaking for the time shown in table 2. Continue dehydrating the sample through a methanol gradient.

Incubate the sample in 100 percent DCM for 1 hour with shaking. DCM Washed samples dry out easily in the air, which would affect the clearness of sample and result in blurry images. It is important to transfer samples quickly into fresh solution to prevent desiccation.

The sample should sink to the bottom of the tube at the end of each DCM wash. Incubate the sample in Dibenzyl ether or DBE overnight with mild shaking to achieve refractive index matching. The sample will eventually become transparent, invisible light.

The next day, after incubating the sample with fresh DBE with mild shaking for 2 hours, store the sample at room temperature in the dark or proceed directly to imaging. To image the sample with a light sheet microscope that is compatible with DBE, mount the agarose embedded sample onto a holder that can prevent movement during imaging. To image the sample with an inverted confocal or two-photon microscope place the agarose embedded sample into a chamber slide with a glass bottom.

Scanning a posterior subcutantious fat pad using a light sheet microscope with low magnification shows the lobular organization of Adipocytes. More detailed information such as the size of the Adipocytes can be revealed by zooming into the regions of interest with higher magnification. A posterior subcutantious fat pad stained with Tyrosine Hydroxylase, a marker for the sympathetic nervous system was imaged by light sheet florescence microscopy.

TH staining revealed structures that appear as large nerve bundles as indicated by the arrow here. Blood vessel innervation as well as dense terminal arborization in the tissue Parenchyma are also revealed by TH staining. Using platelet endothelial cell adhesion molecule or PCAM1 also known as CD31 as a marker to label blood vessels, it is observed that all Adipocytes are in contact with the capillaries throughout the whole tissue supporting the high demand for efficient nutrient and oxygen exchange in Adipose.

After its development, this technique paved the way for researchers in the field of Adipose Biology to explore additional questions such as early tissue morphogenesis and fate mapping of Adipocyte Progenitors. It can also be applied to study the histology of human Adipose Tissue. Don’t forget that working with paraformaldehyde, methanol, dichloromethane and Dibenzyl ether can be extremely hazardous and precautions such as using a fume hood and personal protective equipment should always be taken while performing this procedure.