Kaiser s gelatine

Genomic alterations of human enteric progenitors were evaluated by DNA fluorescence in situ hybridization (FISH) as described recently [22] . Briefly, after preparation of cell metaphases and interphases, cells were denatured, dehydrated and hybridized with multi-colour DNA FISH probe mixtures for 12–16 h at 37°C (ToTelVysionTM, Abbott, Wiesbaden, Germany). These probes bind to the sensitive subtelomeric chromosomal regions which are immediately adjacent to the long (TTAGGG)_n repeats and also contain repetitive stretches of DNA (Figure 1I). After washing, slides were air-dried for 20 min in the dark, incubated with 4′-6-Diamidino-2-phenylindole (DAPI) solution (125 μg/ml; Abbott, Wiesbaden, Germany) for 10 min at room temperature and coverslipped with Kaiser’s gelatine (Merck, Darmstadt, Germany).

Gut tissue was cryo-embedded, sliced and fixed with 4% PFA overnight. Tissue was then incubated in a solution of 3 ml PBS, 1.5 mg nitroblue tetrazozolium (Sigma), 3 mg β-Nicotinamide adenine dinucleotide phosphate (β-NADPH, Sigma) and 1.5 μl Triton X-100 (Sigma) at 37°C for 30 min in the dark (Wallace et al., 2009 (link)). Sections were then washed in PBS and nuclear fast red staining (Vector Laboratories Inc., Burlingame, USA) was performed by incubation with the coloring agent for 20 min at room temperature. The reaction was stopped by washing in PBS. The NADPH-staining was preserved by covering the tissue sections with Kaiser's gelatine (Merck) and cover slips. Samples were visualized and analyzed under morphological peculiarities with Nikon intenselight C-HGFI (Nikon). For quantification, NOS-positive cells in the plexus myentericus region were counted microscopically (25× objective) from 5 non-consecutive longitudinal serial cryosections (16 μm × ~1.5 cm) of each donor (children n = 4, aged donors n = 6).