Goat anti rat secondary antibody

Neutrophil immunohistochemistry was performed using the same protocol to GFP immunohistochemistry above; with primary rat anti-Ly6g antibody and Goat anti-rat secondary antibody (Abcam, Cambridge, U.K.).
Cervical sections were visualized using a x5 objective lens and the epithelium and sub-epithelial stromal areas were identified. Five random fields of view were selected using a x40 objective lens. The numbers of neutrophils were counted per area. Three to five sections were counted per mouse and the number of neutrophils counted per area averaged per mouse.

Time-mated pregnant females were injected subcutaneously with 5-bromo-2′-deoxyuridine (BrdU) 100 mg kg⁻¹ (Sigma Aldrich, USA) at E10.5, E11.5 or E12.5. Embryos were isolated at E15.5 and dissected brain were fixed for 1.5 h in 4% PFA on ice and further processed as for immunohistochemistry. For BrdU staining, sections were incubated with 0.1 M citric acid for 20 min at 100 °C, adjusted to room temperature and rinsed three times in PBS before applying the blocking solution containing 0.25% Triton X-100 (Sigma), 5% donkey serum in PBS (Jackson ImmunoResearch, UK). Sections were incubated with rat monoclonal anti-BrdU antibody (1:500, Abcam) diluted in blocking solution at overnight at 4 °C. The next day, sections were washed three times in PBS before being incubated for 1 h with a goat anti-rat secondary antibody (1:500, Abcam).