Mouse anti cytokeratin 7

Kisspeptin-10 (KP) and its antagonist (p356) were custom-synthesized by EZ Biolabs. p356 is a KP antagonist derived from antagonist p234 [22] (link). The source of all other reagents was Sigma unless otherwise indicated.
Antibodies used for Western blot were rabbit anti-GPR54 (GTX100374, GeneTex), rabbit anti-Kisspeptin (ab80994, Abcam), rabbit anti-β-actin (sc-1616, Santa Cruz), rabbit anti-ERK1/2 and rabbit anti-P-ERK1/2 (9102 and 9106, Cell Signalling). HRP-conjugated anti-mouse and anti-rabbit (Santa Cruz) were used as secondary antibodies. Antibodies used for immunocytochemistry were rabbit anti-GPR54 (R2 1212 serum, EZ Biolabs), mouse anti-cytokeratin-7 (M7018, Dako), mouse anti-vimentin (M0725, Dako), rabbit IgG anti-human KSHV GPCR (Cell Sciences) and mouse IgG HA-Probe (F-7) (sc-7392, Santa Cruz). Cy3-conjugated anti-mouse and Alexa 488-conjugated anti-rabbit (Jackson Immuno Research) were used as secondary antibodies.

To analyze Cytokeratin 7 (Dako) and PTTG1 (Invitrogen) expression in placenta, placental tissues were embedded in OCT compound (Tokyo, Japan, Sakura Finetechnical Co. Ltd), and preserved at -80°C. Two serial sections of 6-μm thick were made of the same tissue specimen, and then fixed in 100% methanol. One section was stained with Hematoxylin and Eosin (H%E) solution. Another section was incubated in protein blocking solution (Dako) at room temperature for 30 min, and then incubated overnight at 4°C with mouse anti-cytokeratin7 (1:100 dilution; Dako) and rabbit anti-PTTG1 (1:100 dilution; Invitrogen), followed by 1 h of incubation at room temperature with Alexa 594-conjugated (1:200 dilution; Invitrogen) and Alexa 488–conjugated secondary antibody (1:200 dilution; Invitrogen). DAPI (Vector Laboratories, Burlingame, CA, USA) was used as a counterstain. Images were analyzed using an EVOS fl microscope (AMG, Mill Creek, WA, USA).