The overall goals of these experiments are to analyze adipocyte homeostasis and differentiation and to investigate the mechanisms of hypoxia and adipocyte apoptosis. This method can help answer key questions in the metabolics field such as obesity and type II diabetes. The main advantage of this technique are that they are basic and inexpensive procedures is that analysis of adipocyte biology in your particular model of studies.
Nicole Hannemann, a Ph.D.student from my laboratory will be demonstrating the procedures. To quantify hypoxia in vivo first determine the body weight of the mouse. Then inject 60 mg per kg of body weight of solid pimonidazole hyrochloride intraperitoneally.
After 45 minutes pin down the limbs of the animal and open the peritoneal cavity. Harvest the left and the right perigonadal fat pads from the cavity and remove the gonadal tissues from the pads. Then weigh the tissue to determine the fat pad per body weight ratio in grams.
To perform histological analysis of the adipocyte homeostasis first deparaffinize the adipose tissue sections with three five minutes washes in xylene, followed by two two minute rehydrations in 100%ethanol and two two minute rehydrations in 96%ethanol. Then wash the sections in distilled water for five minutes and stain them with hematoxylin for 10 minutes at room temperature. At the end of the incubation wash the sections in water for another five minutes followed by a 30 second stain with eosin solution.
Wash the sections as just demonstrated. Then dehydrate the sections in 96%ethanol and 100%ethanol two times each for two minutes. Followed by three five minute xylene incubations.
Then mount the sections with an anhydrous mounting agent. For immunohistochemical analysis of the tissues deparaffinize the sections as just demonstrated, followed by a 30 minute digestion with proteinase K working solution at 37 degrees Celsius. At the end of the digestion rinse the tissues in PBS and block the endogenous peroxidase with 3%hydrogen peroxide in PBS for 10 minutes.
After two subsequent five minute washes in PBS block any non specific antibody binding with 10%serum in PBS. Then incubate the tissues and the antibodies of interest at four degrees Celsius overnight. The next morning rinse the sections with three five minute washes in PBS.
Then incubate the tissues with the appropriate biotinylated secondary antibodies for one hour at room temperature. During the last 30 minutes of the secondary antibody labeling period equilibrate 50 microliters of avidin solution with 50 microliters of biotinylated peroxidase H per section for 30 minutes at room temperature. Then wash the sections two times for five minutes each in PBS and treat the tissues with 100 microliters of avidin biotin ABC complex at room temperature.
After 45 minutes wash the sections two times in PBS and incubate the tissues in the peroxidase substrate solution until the staining reaches the appropriate level of intensity. Now wash the sections for five minutes with distilled water and counter stain the cells with hematoxylin for 10 minutes at room temperature. After counter staining wash the tissues in water again.
Then dehydrate the sections and use an anhydrous mounting agent to mount the samples with cover slips. The sections can then be evaluated under a bright field microscope. Begin by detaching the skin of the adult male mouse from the upper leg, loin and flank pinning the skin as it is removed.
Then harvest the posterior subcutaneous adipose tissue surrounding the inguinal lymph nodes located at the base of the hind legs for culture of the adipocytes. To analyze adipogenic differentiation place the differentiated adipose cell culture under a fume hood and gently wash the cells with PBS. Next fix the cells in two mL of 10%formalyn for 60 minutes.
At the end of the fixation wash the cells in water and treat the culture with two milliliters of 60%isopropanol for five minutes followed by two millilters of oil red O working solution. After five minutes rinse the cells with tap water until the water runs clear. And counterstain the cells with hematoxylin as just demonstrated.
The cells can then be evaluated under a phase contrast microscope at a 100 fold magnification. The lipids of the adipocytes will appear red and the nuclei will appear blue. Here sections of the fat pad from normal and high fat diet mice are shown.
Quantification of the adipocytes'size and area clearly demonstrates adipocyte hypertrophy after six weeks of a high fat diet as evidenced by the increased adipocyte size in the high fat diet treated animals. In mice that had been treated with the hypoxic marker pimonidazole, the increased hypoxic status of the adipose tissue is accompanied by an increase in HIF1 alpha positive adipocytes. Further, tunnel staining of adipose sections from knock out and control litter mates demonstrates that an increase in adipocyte apoptosis correlates with the presence of hypoxia and hypoxic inducible factor 1 alpha expression.
As expected the Hif1a expression in adipocytes increases after 24 hours of hypoxia. More, quantification of Hif target genes by QPCR indicates that the increased expression of Hif1a under hypoxic conditions leads to an increase in Inos mRNA expression. The silencing of Hif1 or 2 alpha by RNA interference, however, rescues the adipocytes from apoptosis under hypoxic conditions confirming the hypoxia sensory regulatory role of Hif alpha in adipocyte apoptosis.
Following these procedures all the assay like determining glucose and insulin resistance can be performed to answer additional questions about metabolic change and adipocyte dysfunction. After watching this video you should have a good understanding of how to perform histologic analysis of adipocyte apoptosis and hypoxia studies.
