Eclipse e800 microscope

Immunohistochemistry was performed as previously described. [13 (link)] Briefly, frozen section slides of the individual groups were incubated with antibodies to smooth muscle actin-α, NFkB p65, IkBα (Cell Signaling, Danvers, MA) and macrophage (MAC397) (Abcam, Cambridge, MA). Images for NFkB p65 and IkBα were captured at 40X magnification with a Nikon E800 Eclipse microscope (Nikon, Tokyo, Japan) at the same exposure in 5 random fields. Image J software was used to quantify mean intensity of respective antibodies. Representative images are included in the manuscript. Images for macrophage antibody (MAC397) were analyzed at 40X magnification with a Nikon E800 Eclipse microscope (Nikon, Tokyo, Japan). Macrophages were counted for each specimen (1 specimen per pig) and totals were divided by specimen area. Representative images are included in the manuscript.

Partial cDNA fragments were amplified using primers specific for rat Xlr5c-like (XM_ 003748905, (5′-CAGACTTGAAAGAGGCCAGG-3′, 5′-TTTGCTGACTGCCAATGAAG-3′) and RT reactions of total RNA samples isolated from ovaries at 12 h post-hCG. The amplified PCR fragment was cloned into pCRII-TOPO Vector. Sequences of the cloned DNA were verified commercially (Eurofins Genomics). Plasmids containing partial cDNA for Xlr5c-like were linearized with HpaI and EcoRV to generate sense and antisense riboprobes, respectively. Linearlized plasmids were labeled using a fluorescein RNA labeling kit (Roche Applied Sciences) and T7 and SP6 RNA polymerase, as appropriate. Ovaries collected from immature rats injected with PMSG or PMSG + hCG were sectioned at 10 μm and mounted on Probe On Plus slides (Fisher Scientific). In situ hybridization analysis was carried out as described previously with a slight modification (Jo et al, 2004 (link)). Briefly, the ovarian sections hybridized with the probes were incubated with anti-fluorescein antibody (Roche Applied Sciences) at 4°C overnight and the signals for Xlr5c-like mRNA were amplified using a TSA™-plus fluorescein kit (Roche Applied Sciences). The sections were counterstained with propidium iodide for 20 min and fluorescent staining specific for Xlr5c-like mRNA was visualized with an Eclipse E800 Nikon microscope under fluorescent optics.