All animal experiments were performed according to Swiss animal welfare laws and were approved by the local veterinary authorities (Kantonales Veterinäramt Zürich; protocol no. ZH269/16). Male, 6- to 8-week-old C57BL/J6-Crl1 mice were purchased from Charles River Laboratories, Inc. (Sulzfeld, Germany). A total of 12 animals were used in this study. All animals were kept at an in-house animal facility with free access to a standard chow diet and water under a standard 12 h light/dark cycle.
Tissue preparation, fixation, decalcification, embedding, and sectioning
Animals were sacrificed by CO2 inhalation. For tissue fixation, the thoracic cavity was opened, and a cannula was inserted in the left cardiac ventricle for transcardial perfusion with 10 ml phosphate buffered saline (PBS), followed by 10 ml of a fixative, i.e., (i) 10% neutral buffered formalin (F; Lucerna-Chem AG, Luzern, Switzerland), (ii) F with 1% acetic acid (FA; Sigma-Aldrich, Steinheim, Germany), (iii) F with 0.2% glutaraldehyde (GA; Sigma-Aldrich), or (iv) FA with 0.2% glutaraldehyde (FGA). The respective fixative used in each protocol is given in Table 1. Then, the animals were decapitated, soft tissues were removed from the outer surface of the skull using a sharp scalpel, and the cranial part of the skull was opened to remove most of the cerebrum, while the cerebellum was left in situ. The tympanic bullae were opened to allow faster penetration of the fixative into the inner ear. The specimens were then immersed in the respective fixative used for transcardial perfusion for 6 h on a countertop shaker. Afterwards, the specimens were dehydrated in a graded series of ethanol solutions (50%, 70%, 95%, and 100%), embedded in paraffin and sectioned at 4 μm using an HM 355S Automatic Microtome (Thermo Fisher Scientific, Waltham, MA, USA). Sections were collected on SuperFrost Plus slides (Thermo Fisher Scientific), dried on a heating plate at 37 °C overnight and stored at room temperature.
Immunohistochemical DAB labeling
Sections were deparaffinized in Histo-Clear (National Diagnostics, Atlanta, GA, USA), rehydrated in a graded series of ethanol solutions (100%, 95%, 70%, and 50%), and rinsed in tap H2O. When required, heat-induced antigen retrieval (HIAR) with pressurized coverslipping of the mounted tissue sections was performed (Table 1) according to a previously established protocol (Eckhard et al. 2019a). All subsequent steps were performed at room temperature. Nonspecific binding was blocked with 1% normal horse serum (NHS) for 15 min, followed by incubation with primary antibodies overnight. The sections were then incubated with biotinylated secondary antibodies for 1 h, followed by incubation with avidin-biotin-HRP complex (Vectastain ABC HRP Kit, Vector Laboratories, Burlingame, CA, USA). Visualization was performed with 3,3′-diaminobenzidine (DAB) (DAB Peroxidase [HRP] Substrate Kit, Vector Laboratories). All incubation steps were performed at room temperature, and each incubation step was followed by rinsing the sections in PBS for 5 min. All primary and secondary antibody combinations utilized in this study are listed in Table 1. Negative controls for all immunolabeling experiments were obtained by omitting the primary antibody or after preabsorption of the primary antibodies with the corresponding commercially available control peptides (for primary antibodies against TRPV5/6, PMCA1/4, NCX2, and SERCA1/2). For nuclear counterstaining, the slices were incubated for 5 s with hematoxylin diluted in distilled water (1:6) and mounted with a permanent mounting medium (VectaMount, Vector Laboratories).
Immunofluorescence double labeling
Deparaffinization, rehydration of the sections, and incubation with primary antibodies was performed as described for DAB-labeling experiments. All primary antibodies used for immunofluorescence double labeling are listed in Table 2. The sections were then incubated with biotinylated antibodies raised in donkey and directed against mouse (1:400; 715-065-151, Milan Analytica AG, Rheinfelden, Switzerland) and Alexa Fluor 594-conjugated antibodies raised in goat and directed against rabbit (1:400; 111-585-144, Milan Analytica AG) for 1 h, followed by incubation with avidin-biotin-HRP complex (Vectastain ABC HRP Kit, Vector Laboratories). Biotinylated tyramine (10 min) and a subsequent second incubation with avidin-biotin-HRP complex were used for signal amplification. Visualization was performed with Alexa Fluor 488-conjugated streptavidin (1:800; Milan Analytica AG) and Alexa Fluor 594-conjugated anti-goat antibodies raised in donkey (1:400; 705-585-003, Milan Analytica AG). The sections were mounted with an aqueous mounting medium containing DAPI for nuclear counterstaining (H-1200, Vectashield, Vector Laboratories).
Images were acquired using either a Leica DMI6000 microscope (Leica, Wetzlar, Germany) or a Leica SP5 confocal laser scanning microscope (Leica) and processed with Adobe Photoshop CS5 software (Version 12.0, Adobe Systems, San Jose, CA, USA).
Quantification of immunolabeled ES epithelial cells
DAB-labeled (DAB+) ES epithelial cells were quantified on light microscopic (× 40 air objective) images of hematoxylin-counterstained tissue sections. Cell counting was performed in at least three non-consecutive sections from two different axial planes of the intraosseous ES (iES) and the extraosseous ES (eES). Each section was divided into ten equally long segments along the longitudinal axis of the ES. The percentage of DAB+ cells among the epithelial cells with hematoxylin-stained nuclei was determined in each segment.
Single- and double-immunofluorescence-labeled ES epithelial cells in the eES were quantified following double immunofluorescence labeling of CaSR with either V-ATPase, TRPV5, or γENaC. The percentages of fluorescence-labeled cells among the epithelial cells with DAPI-counterstained nuclei were determined. From each double immunofluorescence labeling experiment, at least 100 labeled cells were counted, and sections from at least 3 independent specimens were analyzed.
Statistical analyses were performed using Prism software (version 7.0, GraphPad Software). Cell counts are expressed as percentages of the number of epithelial cells in a segment of the ES. Mean values and standard deviations derived from six animals are shown.