Cy3 conjugated goat anti rabbit igg antibody

The cultured cells were washed three times with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde (Sigma-Aldrich) for 30 min. Following inhibition, the cells were initially incubated with primary antibody overnight at 4°C, and then with fluorescein isothiocyanate- or Cy3-conjugated goat anti-rabbit IgG antibody (1:200; Sigma-Aldrich) and 5 μg/ml DAPI (Sigma-Aldrich) at room temperature for 30 min. Then, the cells were thoroughly washed with TBST and viewed through a fluorescence microscope (DMI3000; Leica, Allendale, NJ, USA).

Cells were rinsed in PBS three times and fixed with 4% formaldehyde in PBS for 15 min at room temperature. After that, goat serum was used to block nonspecific binding sites. The cultured cells were then incubated with an MMP-13 antibody (Abcam; 1:200 dilution) in PBS for 8 h at 4°C. After three washes with PBS, each for 3 min at room temperature, the cells were incubated for 1 h with a Cy3-conjugated goat anti-rabbit IgG antibody (1:100 dilution, Sigma–Aldrich, St. Louis, MO, U.S.A.) for 1 h, and counterstained with DAPI. After the final round of three washes, the samples were mounted on slides and examined under a confocal microscope (Nikon Eclipse 80i; Nikon, Tokyo, Japan).

The small intestines and ovaries of mice were fixed for 2 h at 4 °C in a fresh solution of 4% paraformaldehyde (Wako, Japan). The samples were then washed in PBS, incubated overnight at 4 °C in a solution of 30% sucrose, and embedded in OCT compound (Sakura Finetek). The tissue segments were sectioned with a cryostat at 8 μm and stained for AID using a tyramide signal amplification system kit with TSA Plus TMR (PerkinElmer)^{42 (link)}. GDF-9 were assessed by indirect immunofluorescent staining. Immunohistochemical staining was performed using a standard avidin–biotin complex peroxidase method, as described previously^{43 (link)}. The primary antibodies used in this study were as follows: rat monoclonal anti-AID antibody MAID-2 (eBioscience, 14-5959-80)^{44 (link)}, rat monoclonal anti-AID antibody (Merck Millipore MABF63, clone 328.8), rabbit polyclonal anti-GDF9 antibody (abcam, ab93862), and rat IgG2b kappa isotype control (eBioscience, 16-4031-81, clone eB149/10H5). The secondary antibodies used in this study were as follows: Peroxidase AffiniPure F(ab’)₂ donkey anti-rat IgG antibody (H + L) (Jackson Immunoresearch, 712-036-153), Cy3-conjugated goat anti-rabbit IgG antibody (Chemicon, AP132C).