A standardized methodology for identifying and dissecting adipose depots throughout the body is critical for the investigation of differences in the character and function of adipocytes within discrete anatomical niches. The main advantage of this technique is that it is a reliable and accurate method for identifying and excising both common and less studied rodent adipose depots. For the identification and isolation of interscapular BAT, use iris scissors to cut horizontally along the bottom edge of the anterior subcutaneous tissue of a euthanized mouse following the natural border of the depot.
Make two vertical incisions along the lateral edges of the depot following the natural borders of the tissue. Use forceps to carefully flip up the depot to reveal the butterfly-shaped interscapular brown adipose tissue embedded within the white adipose tissue. Then carefully dissect the BAT away from the surrounding WAT.
Store adipose tissues in appropriate containers for downstream analysis. For the identification and isolation of posterior subcutaneous WAT, place the mouse into the supine position and secure the animal's limbs. Use forceps to lift up the skin at the base of the sternum and make a one millimeter skin incision.
Insert one blade of the iris scissors into the cut and make a four to five centimeter midline skin incision beginning at the base of the sternum and ascending to the base of the tail. Next, make two one centimeter horizontal incisions at the top of the initial vertical incision extending laterally from the midline. Use the forceps to carefully peel back the skin from the peritoneal cavity, use the leg to locate the posterior subcutaneous WAT which should remain associated with the skin.
Pin the outstretched skin and completely excise the triangular inguinal WAT depot from the base of the hindlimb ventrally across the groin. For visceral WAT depot identification and isolation, use forceps to lift up the thin peritoneal cavity wall at the base of the sternum and make a one millimeter incision in the tented tissue. Insert one blade of the iris scissors into the cut and make a four to five centimeter descending vertical incision from the top of the peritoneal cavity to the rectum.
Make two one centimeter horizontal incisions at the top and bottom of the vertical incision extending laterally from the midline and use the forceps to peel back the peritoneum to expose the abdominal cavity contents. Then pin the outstretched peritoneum to the dissection pan to harvest the WAT depots contained within the peritoneal cavity. For the identification and isolation of visceral gonadal WAT, locate the gonads and use forceps to lift up the associated gonadal WAT, then use iris scissors to carefully excise the WAT from the gonads.
To excise the perirenal depot, locate the kidney and use forceps to lift up the renal tissue. Pull the kidney to the midline to see a clear division between the perirenal and retroperitoneal depots and excise the WAT directly associated with the kidneys. Then remove the adrenal glands located above the kidneys from the WAT.
For retroperitoneal WAT identification and isolation, lift the kidney to the midline again and use iris scissors to carefully dissect the retroperitoneal WAT from the posterior peritoneal wall. To isolate the popliteal WAT, place the patella against the dissection pan taking care that the popliteal fossa at the back of the knee is facing upward. Use the iris scissors to carefully remove the skin from the base of the hindlimb to the foot.
Use the iris scissors to make a cut at the inferior border of the medial and lateral heads of the gastrocnemius muscle, use the forceps to lift up the muscle to reveal the triangular popliteal depot. Then use iris scissors to excise the depot along the natural tissue borders. Here, the gross anatomical locations of mouse subcutaneous, brown, visceral, and popliteal depots are shown.
The histological characteristics of the subcutaneous, brown, visceral, popliteal, constitutive, and regulated marrow adipose, intramuscular, and infrapatellar adipose depots can be evaluated by standard Hematoxylin and Eosin staining protocols. The adipose tissues isolated using this protocol can be used for a wide variety of experimental endpoints including histological analysis, molecular studies, and gene and protein expression analysis. Field-wide standardization of the identification of diverse adipose depots will undoubtedly help us further our understanding of their molecular and metabolic characteristics and their differential contributions to both local and systematic pathological states.
