Erα mc 20

Adipose tissues were collected from 95 day old mice and fixed overnight in 10% formalin. The Molecular Pathology Core at UT Southwestern Medical Center embedded tissues with paraffin, sectioned and immunostained with PHD3 antibody (Novus, NB100-303) or ERα antibody (Santa Cruz, ERα MC-20, #sc-542).

Immunohistochemistry and immunofluorescence for paraffin-embedded tissues were carried out as described previously.^{24 (link)} Primary antibodies against α-SMA (clone E184, 1 : 50; Millipore, Billerica, MA, USA), CK8 (ks8.7, 1 : 50; Santa Cruz Biotechnology, Dallas, TX, USA), ERα (MC-20, 1 : 50; Santa Cruz Biotechnology) or PR-A (hPRa7, 1 : 50; Labvision, Fremont, CA, USA) were used. Alexa-488- or Alexa-594-conjugated secondary antibody (1 : 200; Molecular Probes, Carlsbad, CA, USA) and 4′, 6-diamidino-2-phenylindole for immunofluorescence were used. Immunofluorescence staining of in vitro colonies was as described previously.^{24 (link)} Briefly, colonies were extracted from matrigel by using PBS-EDTA and then spun to glass slides with a Cytospin4 centrifuge (Thermo Scientific, Kalamazoo, MI, USA). The colonies were fixed with 4% paraformaldehyde, followed by the immunofluorescence staining procedures. Slides were analyzed by confocal laser scanning microscopy (Carl Zeiss, Oberkochen, Germany).