Mouse anti α smooth muscle actin

Immunofluorescence was performed on 5–8 μm cryostat sections of tissues freshly embedded in OCT as described^{29 (link)}. The sections were fixed in 4% paraformaldehyde in PBS and then stained with rat anti-PECAM (1:200, BD Biosciences #557355), mouse anti-α smooth muscle actin (1:200, Abcam #ab7817), rabbit anti-phospho-JNK (1:200, Cell Signaling), rabbit anti-UCP1 (1:200, Abcam #ab10983) and rabbit anti-ERα (1:200, Abcam #ab2746) as described^{29 (link)}. Secondary antibodies, used at a 1:500 dilution, included cy3 donkey anti-rabbit, cy3 donkey anti-mouse, cy3 donkey anti-rat, cy5 donkey anti-rat, cy5 donkey anti-rabbit, cy3 donkey anti-mouse were from Jackson ImmunoResearch. Immunostaining images were collected on a Zeiss LSM500 confocal microscope, an Olympus IX70 inverted microscope or an Olympus upright BX40 microscope. Direct GFP and RFP fluorescence for whole-adipose depots were imaged and photographed with a Zeiss Stemi SV11 microscope. Cryostat sectioning was performed with a Microm HM505 E cryostat. For BrdU staining, the cells, sections or tissues were fixed and washed in H₂O, incubated with 1 N HCl at 37 °C for 45 min, washed in H₂O, incubated in 0.1 M NaBO₄ for 10 min and subjected to immunohistochemistry.