Dapi mounting solution

Colonic tissues were fixed in 10% formaldehyde and embedded in paraffin and then 5-μm sections were obtained. Apoptosis was detected by TUNEL staining using an in situ cell death detection kit (Roche). DAPI mounting solution (Vector, Burlingame, CA, United States) was used for nuclei staining. The results were observed under a light microscope and representative pictures were photographed.

The ileum samples were opened longitudinally, fixed with 10% formaldehyde, and then embedded in paraffin. Thereafter, samples were sliced as sections with 5-μm thickness for TUNEL staining using an in situ cell death detection kit (Roche, Shanghai, China). Nuclei were stained using DAPI mounting solution (Vector, Burlingame, CA, United States). Representative results were collected using a light microscope.

Serum-starved (overnight) cells on cover slips were incubated with nocodazole (10 μM; Sigma) for 4 hours [38 ]. The cells were then washed with serum-free medium (3x) to remove the drug and the cover slips collected at various time intervals. Cells were fixed with 4% paraformaldehyde in PBS for 10 min and then permeabilized with 0.5% Triton-X100 in PBS for 10 min. For immunofluorescence staining, cells were stained with anti-vinculin (Cat No V4505, Sigma), and anti-GATA2 (Novus Biological); secondary antibodies were obtained from Jackson Immuno Research (West Grove, PA). Slides were mounted using DAPI mounting solution (Vector Laboratories, Burlingame, CA) and viewed using a LSM 780 Confocal Microscope (Carl Zeiss Inc.).

One methodological limitation in the fluorometric measurement of telomere length is in the semi-quantitative assessment of fluorescence intensities. To overcome this limitation, we applied a new approach in the investigation of telomere length by creating a conversion factor to calculate TFI into kb length. For this, we used we used five cells lines with defined and stable telomere length (TL) to generate a standard curve to calculate cell type specific TL of each individual (Supplemental Table 2). TL of the appropriate cells was assessed by Southern blot analysis as described previously [65 (link)]. All five cells lines were qFISH analyzed together with tissue of ALS-patients and control individuals, which were not processed with citrate buffer and pepsin digestion. Cells lines were thawed on ice and approximately 1×10⁵ cells were used for TL analysis. Cells were washed, dehydrated and hybridized as described above. Then slides were washed twice for 30 min with formamid wash buffer (70 ml Formamide, 1 ml Tris (1M) pH7.2, 1 ml BSA (10%), 28 ml H₂O) followed by three 5 min washes with TBS-Tween 1% and two 5 min washes with PBS. Then slides were mounted in DAPI mounting solution (Vectashield).