P8825

For PCNA immunohistochemistry, we chose the sections immediately adjacent to the one identified as having the highest number of GnRH3 neurons. These sections were incubated in methanol containing 0.3% hydrogen peroxide to block endogenous peroxidase activity. Next, sections were immunohistochemically stained with mouse monoclonal anti-PCNA antibody (1:3000, P8825, Sigma-Aldrich) diluted in 0.1 M PBS containing 0.03% Triton X-100 and 10% Block Ace and with biotinylated goat anti-mouse IgG (1:500, Vector Laboratories, Burlingame, CA, USA). After incubation with an avidin-biotin-peroxidase-complex (ABC Method kit, Vector Laboratories), the sections were incubated with 0.02% solution of 3, 3′-diaminobenzidine (Wako Pure Chemical Industries) in 0.175 M sodium acetate buffer containing 0.125% nickel chloride (Wako Pure Chemical Industries, Osaka, Japan) and 0.0026% hydrogen peroxide (Wako Pure Chemical Industries).
In every PCNA-stained sections obtained per animal, the number of cells with PCNA-positive nuclei (PCNA-positive cells) were counted in the dorsal and ventral areas (each area, 140 × 140 µm) around the ventricle (Fig. 1a). Average dorsal and ventral densities of PCNA-positive cells per animal were calculated, and mean densities and standard errors of PCNA-positive cells were then determined for control (n = 3) and 11-KT injected (n = 3) animal groups.

Cells were plated on glass coverslips and fixed with 4% paraformaldehyde in PBS for 20 min at room temperature. When indicated, cells were incubated with EdU (5-ethynyl-2′-deoxyuridine). PFA-fixed cells were permeabilized with 0.5% Triton X-100 in PBS for 20 min. EdU was coupled with Alexa Fluor 555 or biotin–TEG azide using click chemistry. Primary antibodies against SMC1 (A300-055A; Bethyl), SMC3 (A300-060A; Bethyl), MCM5 (17967; Abcam), PCNA (P8825; Sigma-Aldrich), and biotin (A150-109A; Bethyl or 200-002-211; Jackson ImmunoResearch) were incubated overnight. Probes from Duolink In Situ PLA Probe Anti-Rabbit PLUS (DUO92002; Sigma-Aldrich) and Duolink In Situ PLA Probe Anti-Mouse MINUS (DUO92004; Sigma-Aldrich) were incubated with coverslip for 60 min at 37°C. For ligation (30 min at 37°C) and amplification (100 min at 37°C), Duolink In Situ Detection Reagents Green (DUO92014; Sigma-Aldrich) was used. The cells were mounted on glass slides with Duolink In Situ Mounting Medium with DAPI (DUO82040; Sigma-Aldrich). Cells were analyzed by fluorescence microscopy, and quantification of PLA signal was performed using CellProfiler software (Carpenter et al, 2006 (link)).

Dissected tails (embryos from 2.7 to 3.2 cm long) or jaws (embryos between 4.9 and 6,6 cm long) were demineralized for 1–2 h respectively in MORSE solution (sodium citrate 10 % and formic acid 20 %) at room temperature prior to dehydratation, embedded in paraplast and cut to 10 μm thickness. PCNA immuno-staining was performed using a primary anti-PCNA dilution at 1:500 (P8825, Sigma). For epitope retrieval, sections underwent microwave-induced heat treatment in Tris EDTA buffer at pH9/tween 20 0.05 % (40 s at 600 W until boiling and then 20 min at 120 W). Cell nuclei were counterstained with Hoechst (Sigma).

The antibodies used in this work were against: H3 (Abcam, ab1791, dilution 1/2000), H2B (Abcam, ab1790, dilution 1/2000), NUPs (Abcam, mab414, dilution 1/100), SMC1 (Bethyl, A300-055A, dilution 1/1000), phosphorylated CHK1 (Cell Signaling, 2341 S), PCNA (Sigma, P8825, dilution 1/2500), DNA polymerase alpha (Abcam, ab31777, dilution 1/500), RPA32^{37 (link)} (dilution 1/500) Geminin, CDT1, ORC2^{56 (link)} (dilution 1/1000), MCM3^{57 (link)} (dilution 1/2000), CDC45^{58 (link)} (dilution 1/1000), ELYS^{9 (link),10 (link)} (dilution 1/50) and CDC7^{59 (link)} (dilution 1/1000).

Gonadal regions from individuals at stage 39 were processed, fixed in Bouin’s solution, embedded in paraffin, and sagittal sectioned at 5 µm. Sections were washed with 0.1 M phosphate-buffered saline (PBS pH 7.4) and blocked in 0.1 M PBS containing 0.5% bovine serum albumin (Sigma-Aldrich) and 0.5% Triton X-100 for 60 min before overnight incubation with primary antibody anti-OLVAS (1:200, rabbit, Abcam 209710) and anti-PCNA (1:200, mouse, Sigma P8825) at 4°C. After incubation, the sections were washed twice in PBS and incubated at RT for 90 min with Alexa Fluor 488-conjugated goat anti-rabbit IgG (Thermo Fisher Scientific, A-11008) and 594-conjugated goat anti-mouse IgG (Thermo Fisher Scientific, A32742) secondary antibodies at a dilution of 1:2000 in PBS. After incubation, sections were rinsed twice with PBS and mounted with Fluoromount mounting medium (Sigma-Aldrich) containing 4',6-diamidino-2-phenylindole (DAPI, 5 µg/ml, Life Technologies).

Protein samples concentrations were determined with a BCA Protein Assay Kit (Thermo Scientific). Equal amounts of protein were loaded onto SDS-PAGE gels. Western blot analysis was performed as previously described [32 (link), 33 (link)]. Proteins were electrotransferred onto PVDF membranes (Millipore). Chemiluminescence was performed using the Luminata HRP substrate (Millipore). Bands were detected either by exposure of the probed membranes to X-ray film or by scanning with a Bio-Rad Universal Hood II running Image Lab software (Bio-Rad). Western blot quantification was performed using ImageJ software.
Antibodies used were as follows: MCM2: ab108935 (Abcam); MCM3: 4012 (Cell Signaling); MCM6: sc-9843 (Santa Cruz Biotechnology); MCM7: ab2360 (Abcam); MCM7: 3735 (Cell Signaling); PCNA: P8825 (Sigma); total p53: 9282 (Cell Signaling); and β-actin: A1978 (Sigma).