For MIF staining, sections (4-im thickness) of paraffin-embedded placenta tissues were cut, deparaffinized, rehydrated, and washed in Tris-buffered saline (TBS; 20 mM Tris-HCl [pH 7.6], 150 mM NaCl). TBS was used for all subsequent washes and for dilution of the antibody. Antigen retrieval was carried out by incubating sections in 10 mM sodium citrate buffer (pH 6.0) in a microwave oven at 750 W for 5 min. For CSF1 immunostaining, frozen tissues were cut (6-im thickness) and fixed in ice-cold acetone.
Sections were rinsed in 3% hydrogen peroxide, to block endogenous peroxidase, and incubated overnight with polyclonal antibodies against MIF and CSF1 at dilutions of 1:300 and 1:50, respectively. The slides were then washed three times with TBS for 5 min, and incubated with peroxidase-labeled rabbit anti-goat antibody (Calbiochem) at a dilution of 1:2000 for 30 min. After washing three times for 5 min in TBS, the sections were incubated in diaminobenzidine tetrahydro-chloride (DAKO, Copenhagen, Denmark) in TBS with 0.01% hydrogen peroxide for 15 min.