Permount solution

After the behavioral testing was completed, then rats were perfused 1X PBS perfusate containing heparin, followed by 800 ml of 4% paraformaldehyde in 0.1 M PBS. The brains were placed fixed brain in 20% sucrose solution and incubate at 4 °C overnight. The brains were cut through the midline with a fresh blade at the level of the PVN. Brain tissue were incubated for 30 min in 0.1 mg/ml nitroblue tetrazolium (Sigma, MO, USA) and 0.1 mg/ml β-nicotinamide adenine dinucleotide phosphate (NADPH) (Sigma, MO, USA) in 0.05 M PBST at 37 °C. And then, the sections were rinsed three times (10 min each) in 0.05 M PBS. The slides were air-dried and then coverslipped with Permount™ solution (Fisher Scientific, CA, USA). The number of NADPH-d positive cells were measured from areas of 200 um² according to the atlas of Paxinos and Watson [30 (link)] under the light microscope (DP-20, Olympus, CA, USA).

All endometrial and caruncle tissues were collected and fixed in 70% diethyl pyrocarbonate (DEPC) ethanol, dehydrated, paraffin-embedded, and sectioned at 10 µm thickness. Representative sections of each endometrial and caruncle tissue paraffin block from all treatment groups were randomly selected and stained with hematoxylin and eosin (H&E; Sigma-Aldrich, St. Louis, MO, USA). The tissues were dehydrated, cleared, covered with Permount solution (Fisher Scientific, NJ, USA), and subjected to histological examination, using an optical microscope.

Immunohistochemical detection of MMPs and TIMPs was carried out on 5 μm tissue sections mounted on siliconized slides. Briefly, paraffin sections were dewaxed with xylene substitute (Polysciences, PA, USA) and rehydrated in a graded series of ethanol. Antigen retrieval was performed by heating at 95 °C in 10 mM sodium citrate (pH 6.0). Endogenous peroxidases were quenched with 0.3 % H₂O₂ in methanol for 5 min at room temperature. After 3 washes in PBS, the slides were incubated in a blocking buffer containing 1 % (v/v) goat serum and 3 % (v/v) horse serum in PBS for 1 h at room temperature. Sections were incubated overnight at 4 °C with antibodies (diluted 1:150 in blocking buffer) against MMP-2 (Abcam), MMP-9 (Santa Cruz), TIMP-2, and TIMP-3. Sections were washed in PBS and incubated with secondary anti-rabbit, (Santa Cruz), anti-mouse (Santa Cruz), anti-goat, or anti-FITC HRP-conjugated (Takara, Osaka, Japan) antibodies (diluted 1:300 in blocking buffer) for 1 h at room temperature, rinsed, and developed for 10 min using the ABC detection kit (Vector, CA, USA); diaminobenzidine (Vector) was used as a substrate for HRP. Sections were counterstained with periodic acid–Schiff reagent and Harris hematoxylin solution containing 4 % (v/v) acetic acid. Tissues were dehydrated, cleared, and covered with Permount solution (Fisher, NJ, USA).

Final placement of the electrodes was monitored online based on implantation depth and verified histologically at the end of the experiments. Rats were anesthetized with isoflurane and transcardially perfused with 0.9% sodium chloride, followed by 4% formaldehyde. The harvested brains were postfixed for 24 h and immersed in 20% sucrose for 2 days. Coronal cryostat sections (40-μm thickness) were mounted with permount solution (Fisher Scientific) on superfrosted coated slides (Fisher Scientific). Images of a whole section were taken by a HP scanner, and microscope images were taken by a Zeiss microscope.

Haematoxylin and Eosin (H&E) staining was performed as previously described (Heim et al., 2014). Briefly, tissue was fixed in 4% paraformaldehyde overnight at 4°C, dehydrated in MeOH, and placed at—20°C. After paraffin embedding and sectioning onto slides, tissue was deparaffinized, stained with H&E, coated with Permount solution (Fisher Scientific), coverslipped, and imaged using an AxioSkop2 microscope and AxioCam CCD camera.