Frontier coated glass slides

RNAScope in situ hybridization was performed using a RNAScope 2.5 Reagent Kit-Red (Advanced Cell Diagnostics, Hayward, CA, USA) according to the manufacturer’s instructions. Detailed procedures for this assay were described in our previous studies [14 (link), 21 ]. Paraffin-embedded ovaries and frozen brains were sliced into 5- and 16-μm-thick sections, respectively, as described in our previous studies [12 (link), 17 ]. Subsequently, sections were mounted on FRONTIER-coated glass slides (Matsunami Glass, Osaka, Japan), heated in boiling Target Retrieval solution for 5 min, rinsed with ethanol, and dried. Next, sections were treated with proteinase at 40°C for 30 min and then incubated with RNAScope probes at 40°C for 2 hr. Subsequently, amplification steps were performed using Amp1–6 solutions provided in the kit. Hybridization signals were detected by reaction with Fast Red substrate for 10 min at room temperature. Subsequently, sections were counterstained with hematoxylin, dried, and coverslipped with EcoMount (Advanced Cell Diagnostics). The RNAScope probe against mouse Esr2 transcripts was provided by the manufacturer (Mm-Esr2; Cat No. 316121; Advanced Cell Diagnostics). Absence of non-specific signals was confirmed using a RNAScope Negative Control Probe [DapB (bacterial gene); Cat. No. 310043; Advanced Cell Diagnostics].

Paraffin-embedded and frozen sections were transferred to FRONTIER-coated glass slides (Matsunami Glass). Subsequently, mounted sections were treated with 0.3% (v/v) hydrogen peroxidase in methanol to quench endogenous peroxidase and then washed three times with PBS. Heat-induced antigen retrieval was performed in 10 mM citrate buffer (pH 6.0) by autoclaving samples at 121°C for 10 min with a High-Pressure Steam Sterilizer LBS-245 (Tomy Seiko, Tokyo, Japan). Next, sections were covered with a mouse-on-mouse blocking reagent (Abcam, Cambridge, UK) for 30 min and then immunoreacted with an anti-human ESR2 monoclonal antibody (PPZ0506; Perseus Proteomics, Tokyo, Japan) at dilutions of 1:100 to 1:64,000 in PBS containing 0.3% (v/v) Triton X-100 (PBST) for 16 hr at 4°C. Immunoreactive signals were obtained using a horseradish peroxidase (HRP) polymer detector (Abcam) and 3,3'-diaminobenzidine tetrahydrochloride (Merck KGaA, Darmstadt, Germany) with 0.01% hydrogen peroxidase. All sections, except for brain, were counterstained with hematoxylin before dehydration and mounting with Permount Mounting Medium (Thermo Fisher Scientific). Immunoreactive images were captured using a BX51 microscope (Olympus, Tokyo, Japan) equipped with a DP73 digital camera (Olympus) or BX53 microscope (Olympus) with a DP21 digital camera (Olympus).