peptide nucleotide probe  (BIO-CAT Inc)

Journal: Journal of Histochemistry and Cytochemistry

Article Title: Cell Type–Specific Quantification of Telomere Length and DNA Double-strand Breaks in Individual Lung Cells by Fluorescence In Situ Hybridization and Fluorescent Immunohistochemistry

doi: 10.1369/0022155418761351

Figure Lengend Snippet: Flow chart of methodological steps. The methods are subdivided in a telomere and gamma-H2AX staining phase and a second, postelution phase, which include the IF staining of specific cell markers. Integration of data from phase 1 and 2 was done by scanning coordinates. Abbreviations: IF, immunofluorescence; CC10, club cell-10; FISH, fluorescence in situ hybridization; aSMA, smooth muscle actin.

Article Snippet: Next, slides were air-dried and dry slides were incubated, using cover glasses, with a telomere-Cy3 peptide nucleotide acid (PNA) probe (2.70 µg/ml, F1002; Panagene, Daejeon, South Korea) which was diluted in a 2× saline-sodium citrate buffer (SSC) (0.3-M NaCl, 0.03-M sodium citrate), 5% dextran sulfate, 50% deionized formamide, and 0.5% Tween-20 hybridization mixture.

Techniques: Staining, Immunofluorescence, Fluorescence, In Situ Hybridization

Journal: Journal of Histochemistry and Cytochemistry

Article Title: Cell Type–Specific Quantification of Telomere Length and DNA Double-strand Breaks in Individual Lung Cells by Fluorescence In Situ Hybridization and Fluorescent Immunohistochemistry

doi: 10.1369/0022155418761351

Figure Lengend Snippet: Antibody and FISH probe elution procedures are essential for the use of multiple fluorophores. Representative images of a patient with pulmonary fibrosis. Z-stacked LSCM pictures of (A) telomeres (red), (B) gamma-H2AX (yellow), and (C) overlay stains containing the AT2 cell–specific pro-Spc marker (green). (D–F) Magnifications of boxed areas in images A–C, respectively. (G and H) Telomere and gamma-H2AX channels after FISH probe and antibody elution, showing complete loss of cell nucleus–specific signal and persistent presence of autofluorescent collagen and elastin fibers. (I) DAPI DNA staining (blue) and slide coordinates were used to determine the location. (J, K) ImageJ Telometer images showing manually encircled cells of telomere and gamma-H2AX analyzed pictures. Both images were mirrored to (L), indicating pro-Spc (AT2) and caveolin-1 (AT1) specific cell markers, to identify cell types. White arrows indicate autofluorescent collagen and elastin fibers. Scale bars represent 10 µm. Abbreviations: FISH, fluorescence in situ hybridization; LSCM, laser scanning confocal microscopy; AT2, alveolar type 2 pneumocyte; DAPI, 4′,6-diamidino-2-phenylindole; AT1, alveolar type 1 pneumocyte.

Article Snippet: Next, slides were air-dried and dry slides were incubated, using cover glasses, with a telomere-Cy3 peptide nucleotide acid (PNA) probe (2.70 µg/ml, F1002; Panagene, Daejeon, South Korea) which was diluted in a 2× saline-sodium citrate buffer (SSC) (0.3-M NaCl, 0.03-M sodium citrate), 5% dextran sulfate, 50% deionized formamide, and 0.5% Tween-20 hybridization mixture.

Techniques: Marker, Staining, Fluorescence, In Situ Hybridization, Confocal Microscopy

Journal: Journal of Histochemistry and Cytochemistry

Article Title: Cell Type–Specific Quantification of Telomere Length and DNA Double-strand Breaks in Individual Lung Cells by Fluorescence In Situ Hybridization and Fluorescent Immunohistochemistry

doi: 10.1369/0022155418761351

Figure Lengend Snippet: Bronchial club and arterial smooth muscle cells identification using CC10- and aSMA-selective immunofluorescence stains. White arrows point to the (A1–A3) row of bronchus epithelial cells and to (B1–B3) cells of the arterial wall. Z-stacked LSCM pictures of (A1 and B1) telomere (red dots) and (A2 and B2) gamma-H2AX (yellow dots). DAPI DNA staining (blue) and cell-type identification of (A3) CC10-positive club cells (white) and (B3) aSMA-positive smooth muscle cells (purple). Scale bar is valid for all images and represents 10 µm. Abbreviations: CC10, club cell-10; aSMA, smooth muscle actin; LSCM, laser scanning confocal microscopy; DAPI, 4′,6-diamidino-2-phenylindole.

Article Snippet: Next, slides were air-dried and dry slides were incubated, using cover glasses, with a telomere-Cy3 peptide nucleotide acid (PNA) probe (2.70 µg/ml, F1002; Panagene, Daejeon, South Korea) which was diluted in a 2× saline-sodium citrate buffer (SSC) (0.3-M NaCl, 0.03-M sodium citrate), 5% dextran sulfate, 50% deionized formamide, and 0.5% Tween-20 hybridization mixture.

Techniques: Immunofluorescence, Staining, Confocal Microscopy

Journal: Journal of Histochemistry and Cytochemistry

Article Title: Cell Type–Specific Quantification of Telomere Length and DNA Double-strand Breaks in Individual Lung Cells by Fluorescence In Situ Hybridization and Fluorescent Immunohistochemistry

doi: 10.1369/0022155418761351

Figure Lengend Snippet: Nucleus surface area but not DAPI intensity is stable between experiments. (A) Significant spearman correlation in AT2 cells of healthy controls between DAPI intensity and nucleus surface area (r = 0.8521, p<0.0001). (B) Examples of one patient where AT2 DAPI intensity significantly differs between two independent experiments on two consecutive tissue sections. (C) Nucleus surface area and (D) unadjusted telomere fluorescence of the same AT2 cells indicate no significant differences between experiments. DAPI intensity, nucleus surface area, and telomere intensity data were obtained from the Telometer plugin of ImageJ. Each dot represents one cell. Medians are indicated with horizontal bars. The p values indicate significant differences (Mann–Whitney tests; ns = not significant). Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; AT2, alveolar type 2 pneumocyte.

Article Snippet: Next, slides were air-dried and dry slides were incubated, using cover glasses, with a telomere-Cy3 peptide nucleotide acid (PNA) probe (2.70 µg/ml, F1002; Panagene, Daejeon, South Korea) which was diluted in a 2× saline-sodium citrate buffer (SSC) (0.3-M NaCl, 0.03-M sodium citrate), 5% dextran sulfate, 50% deionized formamide, and 0.5% Tween-20 hybridization mixture.

Techniques: Fluorescence, MANN-WHITNEY

Journal: Journal of Histochemistry and Cytochemistry

Article Title: Cell Type–Specific Quantification of Telomere Length and DNA Double-strand Breaks in Individual Lung Cells by Fluorescence In Situ Hybridization and Fluorescent Immunohistochemistry

doi: 10.1369/0022155418761351

Figure Lengend Snippet: Telomere and gamma-H2AX fluorescence measurements in AT1 and AT2 lung cells of a patient with a PARN mutation. (A) FISH-stained telomere signals are significantly higher in AT1 than in AT2 cells (p<0.0001). (B) Immunofluorescent staining of gamma-H2AX showed significantly lower signals in AT1 cells compared with AT2 cells (p<0.0001). Medians are indicated with horizontal bars. Mann-Whitney tests were used for comparisons. (C) Significant negative spearman correlation in AT1 and AT2 cells between telomere and gamma-H2AX signals (r = −0.5202, p<0.0001). Each dot represents one cell. Abbreviations: AT1, alveolar type 1 pneumocyte; AT2, alveolar type 2 pneumocyte; PARN, poly(A)-specific ribonuclease; FISH, fluorescence in situ hybridization; DAPI, 4′,6-diamidino-2-phenylindole.

Article Snippet: Next, slides were air-dried and dry slides were incubated, using cover glasses, with a telomere-Cy3 peptide nucleotide acid (PNA) probe (2.70 µg/ml, F1002; Panagene, Daejeon, South Korea) which was diluted in a 2× saline-sodium citrate buffer (SSC) (0.3-M NaCl, 0.03-M sodium citrate), 5% dextran sulfate, 50% deionized formamide, and 0.5% Tween-20 hybridization mixture.

Techniques: Fluorescence, Mutagenesis, Staining, MANN-WHITNEY, In Situ Hybridization