A31572

Immunostaining was performed according to previously described protocols (60 (link)). Briefly, the collected ovaries were fixed with 4% (w/v) PFA for 12 to 24 hours, dehydrated, embedded in paraffin, and cut into 8-μm sections. After deparaffinizing and gradually hydrating the sections, they were put into 0.01% sodium citrate buffer (pH 6.0) and then subjected to microwave antigen retrieval for 16 min. The sections were blocked with 10% donkey serum (Jackson ImmunoResearch) at room temperature for 1 hour, and then the tissue sections were incubated with primary antibodies overnight at 4°C. Next, the sections were thoroughly washed in PBS, incubated with the secondary antibody at room temperature for 1 hour, then stained with Hoechst 33342 (B2261, Sigma-Aldrich) for 1 min. The main primary antibodies used in immunofluorescence are as follows: goat anti-FOXL2 (1:300 dilution; IMG-3228, Novus Biologicals) and DDX4 (1:300 dilution; ab27591, Abcam). Second antibody dilution: donkey anti-goat Alexa Fluor 488 (1:150 dilution; A11055, Invitrogen) and donkey anti-rabbit Alexa Fluor 555 (1:150 dilution; A31572, Invitrogen). Images were acquired on a Nikon Eclipse Ti digital fluorescence microscope, Leica (DMi8), or Andor Dragonfly spinning-disc confocal microscope. The image data were analyzed by the software ImageJ. Image merging was by using the color-merge channels function in ImageJ.

The primary antibodies used for immunohistochemistry were as follows: mouse anti-PV (1:1,000, 235, Swant), rabbit anti-SST (1:2,000, T-4103, Peninsula), mouse anti-RELN (1:1,000, MAB5364, Millipore), rabbit anti-VIP (1:500, 20077, Immunostar), goat anti-SATB1 (1:100, sc5889, Santa Cruz), anti-RFP (1:500, 600–401-379, Rockland), and rat anti-BrdU (1:500, OBT0030G, Accurate Chemicals). The secondary antibodies used for immunohistochemistry, all raised in donkey and used at 1:2,000 dilution, were as follows: anti-mouse Alexa Fluor 488 (A21202, Invitrogen), anti-mouse Alexa Fluor 555 (A31571, Invitrogen), anti-rabbit Alexa Fluor 555 (A31572, Invitrogen), anti-goat Alexa Fluor 488 (A11055, Invitrogen), anti-rat Alexa Fluor 647 (712–606-153, Jackson Immunoresearch), and anti-rat Alexa Fluor 488 (A21208, Invitrogen).

HSFs were fixed in freshly prepared 4% paraformaldehyde for 30 minutes at 4°C and washed three times in PBS for 5 minutes with a 0.1 M PBS blocking solution containing 6% normal donkey serum, 1% bovine serum albumin, and 0.3% Triton X-100 that was applied for 2 hours at room temperature to block nonspecific binding. Primary antibodies against VDR (1:100, 12550; Cell Signaling Technology, Danvers, MA, USA) and Collagen I (1:400, ab88147; Abcam, Cambridge, MA, USA) were diluted in the dilution buffer (containing 3% normal donkey serum, 0.5% bovine serum albumin, and 0.3% Triton X-100 in 0.1 M PBS), which covered the sections during overnight incubation at 4°C. After washing the sections three times with PBS for 5 minutes each, they were incubated with secondary antibodies for 2 hours at room temperature with either donkey anti-rat IgG (H+L) conjugated to Alexa Fluor 488 (1:400, A-21208; Invitrogen) or donkey anti-rabbit IgG (H+L) conjugated to Alexa Fluor 555 (1:400, A-31572; Invitrogen). The secondary antibodies were diluted in the same solution as the primary antibody. Finally, cell nuclei were stained with 4′,6-diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA, USA). The images were captured with a Zeiss LSM 880 confocal microscope (ZEISS, Göttingen, Germany) under a ×20 magnification objective.