Figure 1.
Structures of LNA and 2’ O-methyl RNA monomers (phosphate and phosphorothioate structures) used.
Figure 2.
FISH detection of H. pylori 26695 strain (ATCC 700392) using FAM- HP_LNA/2OMe_PO and HP_LNA/2OMe_PS probes.
FISH analysis was performed by epifluorescent microscopy in smears, using either 50% (vol/vol) formamide and 4 M urea as denaturing agents in the hybridization buffer. Smears without probe were used as negative control (Control) (a). (2B) Average fluorescence intensity from each probe in 4 M urea and 50% formamide (v/v) buffers; fluorescent signal intensity is expressed in arbitrary fluorescent units (AFU) and was quantified using the by ImageJ software. All images were acquired at equal exposure conditions. Original magnification: 1000x.
Figure 3.
FISH detection of H.pylori by imaging flow cytometry.
FAM labeled 2OMe/LNA probes were analysed in 50% (v/v) formamide buffer and in 4M buffer. A) Representative histograms of the green fluorescence intensity of FAM-labeled HP_LNA/2OMe_PO, HP_LNA/2OMe_PS probes and controls. B) Quantification of the mean fluorescence intensity of each probe in two independent experiments obtained by flow cytometry. C) Representative images of individual H. pylori with different morphologies. The population identified as individual H. pylori bacterium by FISH analysis was manually examined and individual cell events were identified. Individual bacterial cells, shown by Brightfield images (BF, left column) and green fluorescence images (SG, right column).
Figure 4.
FISH detection of H.pylori in paraffin-embedded sections of gastric biopsies, using 2’-O-methyl/LNA FISH detection conditions.
(A) Detection of H. pylori using the HP_LNA/2OMe_PS probe in a histological slide of a gastric biopsy specimen of an infected patient; (B) Experiment control performed in parallel using the HP_LNA/2OMe_PS probe in a histological slide of a gastric biopsy specimen of an non infected patient. Arrows indicate the presence of H. pylori infecting the gastric mucosa. All images were taken at equal exposure times Scale bars: 10 µm. Original magnification of ×1000.