ompany, The Netherlands) transmission electron microscope. The quantity lysosomes in PKC Synonyms thyrocytes was analyzed on TEM micrographs manually, whilst their diameter was measured by using Windows based ImageJ (Image J, Version 1.49j). Measurements had been performed on 10 thyrocytes per group. 2.4. Immunohistochemistry (IHC) and Immunofluorescence (IFC) Immediately after tissue deparaffinization, endogenous peroxidase activity was blocked by incubation of sections with 0.3 hydrogen peroxide in methanol for 15 min. Then, thyroid sections were exposed to heat-induced antigen retrieval to unmask target antigens. Slides had been placed inside a container, covered with 100 mM sodium citrate buffer (pH 6.0), and heated within a microwave oven at 750 W for 3 7 min. Reduction of nonspecific background staining was accomplished by incubation with typical porcine serum (code no. x0901, Dako, Denmark), diluted 1:10 for 45 min. Details on antibodies applied is summarized in Table 1. For analysis of thyroidspecific proteins, the antiserum directed against human thyroid peroxidase (TPO), thyroglobulin (Tg), and sodium iodide symporter (NIS) had been applied overnight at four C (Table 1). For immunodetection of vitamin D-metabolizing enzymes and VDR, antiserum directed against every single protein was applied overnight at 4 C (Table 1). Secondary antibodies, anti-mouse or anti-rabbit HRP-labeled antibodies, had been applied for 1 h at room temperature. All washes and dilutions have been performed employing 0.1 mol/L PBS pH 7.two.Int. J. Mol. Sci. 2022, 23,four ofTable 1. List of major and secondary antibodies used in IHC/IFC staining. Name TPO NIS Tg CYP24A1 VDR CT Anti-mouse, HRP labeled Anti-rabbit, HRP labeled Manufacturer Santa Cruz, Italy Acris, Germany Dako, Denmark Santa Cruz Biotech Inc., Italy Abcam, UK Dako, Denmark Abcam, UK Dako, Denmark Cat. Number sc-376876 EUD4101 A0251 sc-66851 Ab 3508 A576 Ab6820 P0399 Origin Mouse Rabbit Rabbit Rabbit Rabbit Rabbit Donkey Swine Dilution 1:400 1:600 1:500 1:100 1:1000 1:300 1:200 1:To confirm that the observed staining isn’t triggered by non-specific interactions from the antibody using the tissue (unfavorable handle) in case of VDR and CYP24A1, the principal antibody was substituted with an “irrelevant primary antibody”. Irrelevant major antibody for this goal was polyclonal rabbit anti-rat beta-LH (obtained from Dr. A. F. Parlow, National Hormone Peptide System, Harbor-UCLA Health-related Centre, USA). It is actually not expressed in the thyroid, has the identical isotype as the distinct key antibodies (polyclonal rabbit IgG), and was applied at the exact same concentration. To handle the background staining, the key antibodies had been substituted with phosphate-buffered saline (PBS). Parathyroid glands served Met Purity & Documentation because the positive control of IHC staining. Hematoxylin was made use of as counterstain, and slides had been then mounted in DPX medium (Sigma-Aldrich, Barcelona, Spain). Digital photos with the thyroid sections have been created on a DM RB Photomicroscope with a DFC 320 CCD Camera (Leica, Wetzlar, Germany). For double-immunohistochemical labeling of calcitonin (CT) and CYP24A1 (Table 1), Tyramide signal amplification kit with HRP oat anti-rabbit IgG and Alexa Fluor568 tyramide (cat. no. T20924; Invitrogen, Waltham, MA, USA) was employed in accordance with manufacturer’s instructions. To avoid false colocalization making use of two rabbit antibodies, we applied the microwave remedy described by [31]. In short, soon after overnight immunostaining of CT and following incubation with goat anti-rabbit Alexa Flour 488, sections were ri