Our lab is focused on establishing supraclavicular brown adipose tissue or scBAT as a valuable depot for brown fat analysis due to it's anatomical similarities to human brown adipose tissue. This protocol is aimed at increasing accessibility of scBAT research. This depot may be intimidating due to its small size and proximity to large veins in the neck, so it is imperative to provide a simple method of extracting and processing scBAT.
Our methodology stands out firstly by the use of a dissecting microscope when extracting scBAT. This increases the precision and extracting efficiency, which are both critical when working with such a small tissue depot. Secondly, we homogenized the frozen scBAT samples before doing gene expression analysis to increase the RNA yield to compensate for such a low quantity of available tissue.
We are currently focus on defining the genetic lineage of scBAT, identifying the transgressional networks regulating its development, and developing an ex-vivo model for exploring its physiological function in humans. To begin, place the mouse carcass on the work bench. Spray the ventral neck of the mouse with 70%ethanol to minimize fur contamination.
Make an approximately 1.5 centimeter long bilateral incision along the clavicles. From the ends of bilateral incisions, cut about one centimeter longitudinally towards the ears, creating a squared U-shaped incision around the neck. Place the mouse under a dissecting microscope and bring the exposed neck into the focus.
Using forceps, lift the incised skin sections to expose the salivary glands. Now, using surgical scissors and forceps, disconnect the tissue connecting the salivary glands to the surrounding clavicle area and each other. Lift the salivary glands to expose the supraclavicular brown adipose tissue or scBAT depots.
Observe the BAT along the external jugular vein, which is possibly connected to the underside of the salivary glands. Hold the target adipose tissue with forceps and gently peel it away from the salivary glands. After removing adipose tissue, continue detaching the scBAT from the external jugular vein and the neck musculature until the depots on either side of the neck are fully removed.
Inspect the dissected scBAT sample and use forceps to remove any remaining connective tissue. Begin by placing the glass scintillation vial containing the mouse's scBAT in PBS onto the workbench. Replace the PBS with 10 milliliters of cold, 4%paraformaldehyde.
Place the vial on a nutater, rocking at four degrees Celsius overnight to fix the scBAT. The next day, replace the paraformaldehyde solution with 10 to 15 milliliters of PBS. Place the vial on a nutater and rock for 30 minutes at room temperature.
Replace the PBS with 0.85%saline, and continue rocking for another 30 minutes. Then replace the saline with 10 milliliters of 70%ethanol, 0.85%saline solution. Store the vial in a four degree refrigerator overnight or until ready for paraffin and bedding.
The next day, remove the 70%ethanol saline mixture from the vial and add 10 to 15 milliliters of 95%dehydrant alcohol. Rock the vial on a nutater at room temperature for one hour. After the second wash, add 10 to 15 milliliters of 100%dehydrant alcohol, and continue rocking on a nutater for one hour.
To clear scBAT for embedding, add 10 milliliters of toluene to the vial and continue rocking on a nutater until scBAT is transparent. Then embed the scBAT in paraffin wax. Section the scBAT with a microtome and proceed for hematoxylin and eosin staining.
Hematoxylin and eosin staining confirmed that scBAT comprises tissue structures characteristic of brown adipose tissue, including numerous small multilocular adipocytes in both postnatal and adult mice. After dissecting scBAT from the euthanized mouse, immediately put the tissue into a micro centrifuge tube. Poke a hole in the top of the tube with a sterile needle and snap freeze in liquid nitrogen.
Soak a pestle and thin spatula in liquid nitrogen. Pour the snap frozen tissue sample from the micro centrifuge tube into the mortar. Add a small amount of liquid nitrogen to the mortar and thoroughly grind the tissue with the pestle until completely pulverized.
Use the thin spatula to scrape and transfer the pulverized tissue back into the micro centrifuge tube. Once all tissue is transferred, add 500 microliters of RNA isolation solution to the tube and sonicate for 30 seconds using a Pellet pestle motor. Next, use a syringe to pipette up and down 10 times to further lyse the tissue, ensuring no solid particles are visible.
Add 250 microliters of chloroform and vortex occasionally during a five minute room temperature incubation. Centrifuge the samples at 21, 000 G for 20 minutes at four degrees Celsius. Transfer the top aqueous layer into a new tube and add an equivalent amount of 70%ethanol.
Transfer 500 microliters of the lysate into a binding column. Centrifuge at 18, 000 G for 60 seconds, and discard the filtrate. For reverse transcription, add 0.5 to one microgram of RNA diluted in DEPC-treated water to a PCR tube strip.
Then dilute the CDNA to 250 nanograms per microliter using DEPC-treated water, and set up the quantitative PCR. The expression levels of marker genes involved in mediating scBAT function were relatively similar between scBAT and interscapular BAT.
