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. 2019 Aug 7;14(8):e0220636. doi: 10.1371/journal.pone.0220636

Fig 4. The intensity of apoptosis and proliferation in the gastric tissue of guinea pigs infected with H. pylori.

Fig 4

(A) Level of apoptosis was evaluated in the gastric tissue of guinea pigs noninfected or infected with H. pylori after 7 and 28 days from inoculation. (i) Fluorescence intensity of apoptotic cells, detected in the gastric tissue using the TUNEL assay and counterstained with DAPI was measured using the Image J software version 1.48v (National Institute of Health, United States). (ii) Representative images of gastric tissue sections stained with the TUNEL assay, counterstained with DAPI and photographed in a fluorescence microscope (Axio Scope A1, Zeiss, Germany) at wavelengths: 550 nm (excitation) and 590 nm (emission) for TUNEL and 345 nm (excitation) 455 nm (emission) for DAPI. (B) Level of intensity of anti-apoptotic Bcl-xL in the gastric tissue of guinea pigs noninfected or infected with H. pylori after 7 and 28 days from inoculation. (i) Fluorescence intensity of Bcl-xL in the gastric tissue was measured using the Image J software version 1.48v (National Institute of Health, United States). (ii) Representative images of Bcl-xL in the gastric tissue photographed in a confocal microscope (LeicaTCS SP). DAPI was used for nuclear staining. (C) Level of intensity of cleaved pro-apoptotic caspase 3 (CC3) in the gastric tissue of guinea pigs noninfected or infected with H. pylori after 7 and 28 days from inoculation. (i) The fluorescence intensity of CC3 in the gastric tissue was measured using the Image J software version 1.48v (National Institute of Health, United States). (ii) Representative images of CC3 in the gastric tissue photographed in a confocal microscope (LeicaTCS SP). DAPI was used for nuclear staining. (D) Level of Ki-67 (proliferation marker) in the gastric tissue of guinea pigs noninfected or infected with H. pylori after 7 and 28 days from inoculation. (i) Cell proliferation expressed as a labelling index (LI %), indicating the proportion of Ki-67 positive cells (brown labelled-cells) to all epithelial cells, counted using the Image J software program. (ii) Representative images of gastric tissue stained for the Ki67. Black arrows show Ki-67 positive cells. Five animals per group for control and, 10 animals per group for infected individuals (n = 5+5) were examined. The results are presented as median values of four independent experiments performed in triplicates for each experimental variant. Statistical analysis was performed in the nonparametric U Mann-Whitney and Kruskal-Wallis tests. Statistical significance *P < 0.05, **P < 0.01, ***P < 0.001 was obtained for H. pylori infected animals (7 and 28 days after inoculation) vs control animals; H. pylori infected animals 7 days vs animals 28 days from the last inoculation.