Rat anti pecam

Embryos and tissues were fixed in 10% formaldehyde overnight, dehydrated in 100% ethanol, and embedded in paraffin. 8 µm thick sections were used for hematoxylin eosin, Alcian blue and immunohistochemistry staining. Klf2 in situ hybridization was performed as previously reported (Lee et al., 2006 (link)). The following antibodies were used for immunostaining: rat anti-Pecam (1:500, BD PharMingen), rabbit anti-Versican (1:200, Millipore), rabbit anti-DPEAAE (1:200, Pierce-Antibodies).

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.