2.6. Immunohistochemistry

Placenta sections were dewaxed and rehydrated through an ethanol gradient elution, followed by overnight incubation with anti-CD31 (BD Biosciences Pharmingen, San Diego, CA, USA). After washing in TBS, tissues were incubated with the corresponding secondary antibody for 2 h at RT, followed by a reaction with diaminobenzidine (DAB) and counterstained with hematoxylin. A digital tissue biopsy scanner was used to acquire images of the sections. ImageJ (version 1.48) was used to quantify the CD31 integrated optical density (IOD).