For permeabilization and fixation of bacteria, 30 μl of 4% paraformaldehyde (wt/vol) were placed in the wells with care to cover the entire surface, followed by 50% (vol/vol) ethanol for 10 minutes each, and then allowed to air dry. Approximately 20 μl of hybridization solution containing a mixture of the four probes were added to the fixed smears, which were then covered with coverslips and incubated for 1 hour at 70°C. Each 1 ml of hybridization solution contained 200 nM of the probes mixture, 10% (wt/vol) dextran sulphate, 10 mM NaCl, 30% (v/v) formamide,

0.1% (wt/vol) sodium pyrophosphate, 0.2% (wt/vol) polyvinylpyrrolidone, 0.2% (wt/vol) FICOLL, 5 mM disodium EDTA, 0.1% (vol/vol) ICG-001 in vitro Triton X-100 and

50 mM Tris-HCl (all from Sigma-Aldrich, Sintra, Portugal, except disodium EDTA that was from Pronalab, Lisbon, Portugal). Subsequently, the slides were transferred to a Coplin jar containing learn more prewarmed (70°C) washing solution, that ABT-888 order consisted of 5 mM Tris Base, 15 mM NaCl and 1% (vol/vol) Triton X-100 (all from Sigma-Aldrich, Sintra, Portugal), where the coverslips were carefully removed. The washing step was carried out for 30 minutes at 70°C. The slides were allowed to air dry and mounted with one drop of mounting oil and covered with a coverslip. Specificity and sensitivity of PNA probes After optimizing hybridization conditions, experiments with the PNA-FISH were performed on the 33 available strains in order to confirm the practical specificity and sensitivity of the probes. These results were compared with the gold standard susceptibility culturing test (E-test) and with the presence/absence of mutations in the 23S rRNA gene. Validation of the testing protocol in gastric biopsy slides for clinical application To validate the method in the stomach tissue, thirty nine paraffin-embedded gastric biopsy specimens from patients with known resistance antibiotic profile by antibiogram were used. The study was in accordance with the institutional ethical standards. Informed

consent Clomifene was obtained from the patients. Three-micrometer thick paraffin cuts were deparaffinized and rehydrated in xylol and ethanol based on a protocol previously described [21]. Sections were emerged in xylol (Fisher Chemical, Leicestershire, U.K.) three times (firstly for 15 minutes, and then twice for 10 minutes each), absolute ethanol (Panreac, Barcelona, Spain) (twice for 7.5 minutes each) and ethanol decreasing concentrations (95%, twice for 7.5 minutes each; 80%, 10 minutes; 70%, 10 minutes; 50%, twice for 15 minutes each). Finally sections were immersed in 1% (vol/vol) Triton X-100 (Sigma-Aldrich, Sintra, Portugal) solution for 20 minutes at 70°C. Histological slides were then allowed to air dry and the hybridization protocol previously described for smears, with the exclusion of the fixation step, was used.