Computational protocol: The failing human heart is characterized by decreased numbers of telocytes as result of apoptosis and altered extracellular matrix composition

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Protocol publication

[…] The tissue samples were mounted in Tissue-Tek® O.C.T.™ compound (Sakura Finetek, Tokyo, Japan) and cryosections 5 μm thick were prepared. Cryosections were air-dried and fixed for 10 min. in either acetone (−20°C), 4% paraformaldehyde (room temperature) or Carnoys' solution (room temperature). After washing in PBS sections were incubated with 1% bovine serum albumin for 30 min. to block non-specific binding sites. After rinsing in PBS, the samples were incubated overnight with primary antibodies against c-kit (catalogue number A4502 and clone 104D2; Dako, Clostrup, Denmark), Ki67 (clone MIB-1; Dako) and collagen I (Clone COL-1; Sigma-Aldrich, St Louis, MO, USA). Antibodies against carboxyterminal telopeptide (ICTP), N-terminal propeptide I (PINP) and N-terminal propeptide III (PIIINP) were prepared and used as previously described –. Antimouse or anti-rabbit IgG-conjugated with Cy3 or Cy2 (Jackson Immunoresearch Laboratories, West Grove, PA, USA) served as detection systems in single or double immunolabellings. The nuclei were stained with 1 μg/ml 4,6-diamidino-2-phenylindole (DAPI; Molecular Probes, Leiden, the Netherlands). F-actin was fluorescently stained using FITC- (Sigma-Aldrich) and Alexa633-conjugated phalloidin (Molecular Probes). Negative controls were obtained by omitting the primary antibody, in an otherwise similar protocol. The terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) reaction was performed as previously described . The samples were examined by confocal laser microscopy using Leica TCS2 and Leica TCS8 microscopes (Leica Microsystems). Digital images were further processed for 3D-reconstruction using Imaris® 7.4.2 software (Bitplane AG, Zürich, Switzerland). [...] Measurements of immunofluorescence for collagen I and markers of collagen metabolism were carried out using a ×40 Planapo objective and a Leica (Leitz DMRB) fluorescent microscope (Leica Microsystems). Cryosections from at least two different tissue blocks in each case were used. For quantitative analysis, all sections were immunolabelled simultaneously using identical dilutions of primary and secondary antibodies and other reagents. Immunofluorescent images were obtained under identical parameters of imaging, zoom, objectives and fluorescence power. Sections exposed to PBS instead of primary antibodies served as negative controls. Image acquisition settings were standardized for all groups to ensure that the image collected demonstrated a full range of fluorescence intensity from 0 to 255 pixel intensity level and were kept constant during all measurements. For each patient, at least 10 random fields of vision were analysed for the quantity of the collagen markers investigated using analysis software (Leica Microsystems) and NIH ImageJ program (http://rsb.info.nih.gov/ij/). The area occupied by different collagen markers was calculated as percentage of collagen I or of collagen markers labelling per tissue area.To quantify the number of TCs, we used the positive expression of c-kit of cells having a DAPI-positive nucleus and thin prolongations. C-kit-positive prolongations without a DAPI-positive nucleus were considered Tps. Cryosections from at least two different tissue blocks and from ten random fields in each case were used and the number of TCs and Tps were calculated per 1 mm2 myocardial area. The number of proliferating and apoptotic TCs was expressed as absolute numbers of, respectively, Ki67- and TUNEL-positive cells per 1000 TCs. […]

Pipeline specifications

Software tools Imaris, ImageJ
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Homo sapiens
Diseases Aortic Valve Stenosis, Heart Failure, Cardiomyopathies