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In situ hybridization (ISH) was performed on brain tissue sections to examine the expression of D2R and D3R mRNA according to the manufacturer’s protocol (ACD Inc, CA). Results indicated tiny purple‐reddish granules in the striatum under optical microscopy. After mRNA detection, some slides were blocked with 10% BSA, incubated with the primary antibody (Abcam, MA) for postsynaptic density protein 95 (PSD95), and the corresponding secondary antibody (Sigma, CA). Samples were labeled with a diaminobenzidine (DAB) kit (ThermoFisher, TX), and counterstained with hematoxylin (Sigma, CA), and finally analyzed with Image J software.

Tissue microarrays were purchased from US Biomax, Inc. (Rockville, MD), which contain human normal breast tissues and breast cancer tissues. The immunohistochemistry (IHC) staining was performed as previously described (Su et al., 2014 (link); Zhou et al., 2014 (link)). Briefly, after dewaxing with xylence and dehydration with gradient ethanol, the tissue microarrays were incubated with antigen retrieval buffer (BD Biosciences) for 1 hr at 95°C, followed by treatment with 3% hydrogen peroxide (Sigma) for 10 min. Specimens were blocked with 5% defatted milk for 1 hr at room temperature, and incubated with anti-human-Gpr132 antibodies (Sigma; RRID:AB_10745673; 1:100) overnight at 4°C and then with HRP-conjugated secondary antibodies (1:200) for 30 min at room temperature. Immunostaining was performed using a diaminobenzidine (DAB) kit (Thermo scientific, Waltham, MA). The expression levels of Gpr132 were scored in a blind fashion semi-quantitatively according to the staining intensity and distribution using the immunoreactive score as described previously (Su et al., 2014 (link); Zhou et al., 2014 (link)). Briefly, the IHC score = staining intensity (negative = 0; weak = 1; moderate = 2; and strong = 3) × percentage of positive cells (0% = 0; 0–25% = 1; 25–50% = 3; and 75–100% = 4).

To evaluate the differentiation of hematopoietic precursor cells to osteoclasts, bone slices having osteoclasts were stained with a TRACP-kit (Sigma) at +37 °C according to the manufacturer’s instructions. The nuclei were stained with the DNA-binding Hoechst 33258 fluorochrome (Sigma) for 10 min at room temperature. TRACP-positive cells having three or more nuclei were considered as osteoclasts and were counted from bone slices using a fluorescence microscope (Zeiss AX10; Carl Zeiss Ltd., Hertfordshire, England) with a 20×/NA 0.5 objective (Zeiss).
After counting the osteoclasts, the cells were removed from the bone slice by gentle scraping and the slices were dyed with peroxidase-conjugated WGA-lectin (WGA; 20 µg/ml) for 30 min at room temperature. Slices were washed 3 times with PBS and counterstained using a diaminobenzidine (DAB) kit according to the manufacturer’s instructions (Invitrogen, CA, USA). The total area of resorption pits was measured by acquiring images of each bone slice with a fluorescence microscope (Zeiss AX10), 10×/NA 0.25 Objective (Zeiss) and QImaging Retiga 4000R camera with a digital image analyzer (MCID Core 7.0, Ontario, Canada). Ranges of interest (ROIs) were drawn for each resorption pit and analyzed with FIJI (ImageJ) software^{40 (link)}.