Mowiol 4 88

For fluorescence images, fresh tissue biopsies were snap-frozen in Tissue-Tek (VWR) immediately after removal. Serial 12 μm cryosections were prepared with a microtome and fixed with ice-cold acetone for 10 minutes. Sections were mounted using Mowiol 4–88 (Roth) and phase contrast and fluorescence micrographs were taken on an ApoTome.2 (Zeiss) equipped with a Plan-Apochromat 63×/1.40 Oil DIC M27 objective and an EC Plan-Neofluar 10×/0.30 Ph 1 objective.
For histological stainings, fresh tissue biopsies were fixed over night using 4% formaldehyde and embedded in paraffin. 5 μm sections were prepared with a microtome and stained with hematoxylin-eosin. Micrographs were taken on an Eclipse 80i microscope (Nikon).

The following chemicals were used: Dulbecco’s MEM medium (#F0435), FBS superior (#S0615), L-glutamine (#K0282), penicillin (100U/ml)/streptomycin (100U/ml) (#A2212), trypsin/EDTA solution (#L2143) (Biochrom, Berlin, Germany), MEM non-essential amino acid solution (100x) (#M7145, Sigma-Aldrich, Steinheim, Germany), Gibco^® HAT supplement (#21060-017, Thermo Fisher, Dreieich, Germany),CaCl₂*2H₂O (#22322.295), D-glucose (#101174Y), dimethyl sulfoxide (DMSO) (#83673.230), HEPES (#441476L), potassium chloride (#26764.230), and sodium hydroxide (#28244.295) (VWR Chemicals BDH Prolabo, Leuven, Belgium), sodium chloride (#1064041000, Merck, Darmstadt, Germany), D-luciferin (beetle) monosodium salt (#E464X), HaloTag^® Alexa Fluor^® 488 Ligand (#G1001, Promega, Madison, USA), Dynasore (#2897, Tocris Bioscience, Bristol, UK), Hoechst33342 (#1399, Invitrogen, Eugene, USA), Mowiol 4-88 (#0713, Carl Roth GmbH + Co. KG, Karlsruhe, Germany), Paraformaldehyde (PFA) (#18814, Polysciences Inc., Warrington, USA).
Odorants were purchased from Sigma-Aldrich (Steinheim, Germany), Alfa-Aesar (Karlsruhe, Germany) and Chemos GmbH (Regenstauf, Germany) (Table S1).

Bioprinted constructs were fixed in 3.7% formalin (Sigma), embedded in paraffin, and sliced into 10 μm thick sections. After deparaffinization, antigen retrieval was performed in Tris-EDTA (10 mM Tris Base, 1 mM EDTA, pH 9.0) at 95 °C for 30 min. Cells were permeabilized using 1% Triton-X-100 for 15 min, and blocking was performed for 30 min using 5% goat serum. For the characterization of nuclear damage, γH2AX (anti-γH2A.X (phosphor S139), Abcam, ab11174, 1:1000, Berlin, Germany) was used. For the cellular characterization of epithelial cells, pan-cytokeratin (anti-pan Cytokeratin, abcam, ab27988, 1:250) was used. This was followed by incubation with corresponding secondary antibodies (Alexa Fluor 546- or 488-conjugated anti-rabbit or anti-mouse IgG(H+L) (A11005, Thermo Fisher Scientific, Waltham, MA, USA; 1:2000). Nuclear counter-staining was performed with DAPI (Sigma), and slides were mounted in Mowiol 4–88 (Roth). The slides were analysed by fluorescence microscopy (Zeiss Observer, Z1 microscope, Carl Zeiss, Zeiss, Germany).

MLO-Y4 cells or primary osteocytes (PO), cultured on collagen-coated coverslips, were washed with PBS and then fixed in 4% PFA for 12 min. Next, fixed cells were washed three times with PBS, permeabilized with 0.15% Triton X100 in PBS for 10 min and washed three times with PBS. After blocking for 30 min, the cells were incubated with primary anti-GαQE antibody for 1 h and after washing, the cells were incubated with secondary antibody together with 565-phalloidin (Sigma Aldrich, Taufkirchen, Germany) for 1 h. The nuclei were stained with DAPI. After mounting, the cells with Mowiol 4-88 (Carl-Roth, Karlsruhe, Germany) microscopic pictures were obtained.
To ensure the successful isolation of primary osteocytes, E11-staining was performed. Therefore, the cells were fixed at day 7 after isolation with 4% PFA for 10 min, washed two times with PBS, blocked with 10% goat serum for 45 min at room temperature and incubated with the primary anti-podoplanin antibody (Santa Cruz Biotechnology, Heidelberg, Germany) at 1:50 in PBS plus 3% goat serum overnight at 4 °C. The secondary antibody Alexa Fluor 488-labeled goat anti-hamster IgG (Molecular Probes, Eugene, OR, USA) was used at 10 µg/mL for 45 min at room temperature. Nuclei were stained with DAPI and afterwards mounted with Mowiol 4–88.

To stain GlyR surface receptors, GlyRα1 variants, and pDsRed-Monomer-Mem [Takara Bio (formerly Clontech), Mountain View, CA, United States] were co-transfected into HEK293 cells. All steps were performed at room temperature. After fixation for 20 min with 50 μl 4% paraformaldehyde, 4% sucrose solution, cells were washed three times with PBS and blocked for 30 min with 5% (v/v) goat serum in PBS. Afterward, cells were incubated for 1 h with the GlyRα1-specific primary antibody mAb2b (1:500 in blocking solution; epitope amino acids 1–10 of mature GlyRα1; Synaptic Systems, Göttingen, Germany). Cells were washed three times with PBS and incubated for 45 min with the secondary Alexa488-coupled goat-anti-mouse antibody (1:500 in blocking solution; Dianova, Hamburg, Germany). Then, cells were washed three times with PBS, incubated for 5 min with DAPI (1:5000 in PBS; Thermo Fisher Scientific, Waltham, MA, United States) and mounted on glass slides with Mowiol 4-88 (Carl Roth, Karlsruhe, Germany). Imaging was performed using an Olympus IX-81 inverted fluorescence microscope (Olympus, Tokyo, Japan).