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Mouse organs were examined histopathologically by veterinary pathologists (MR, HP, LM) using formalin-fixed, paraffin-embedded sections stained with hematoxylin and eosin (H&E).
After perfusion, xenograft tumors and mouse organs were removed and homogenized.
Mouse organs were fixed in 4% paraformaldehyde overnight and embedded in paraffin.
To assess the overall ratios of ERM protein expression, various mouse organs were analyzed by western blot.
Mouse organs were also fixed in 4% paraformaldehyde and processed as above.
Mouse organs were excised and stored frozen at −80°C in sucrose until analysis.
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Although bioaccumulation of ACPNs has not been studied in particular, the distribution of HANs in mouse organs was studied via intravenous administration.
RNA from mouse organs was prepared as previously described using a single step isolation protocol [51].
The infection of different mouse organs was monitored by demonstrating the presence of bacterial DNA by nested PCR (Table S1 and S2).
These results demonstrate that the failure of the mutant to grow and persist in mouse organs is due to the mutant's inability to withstand oxidative and nitrosative stresses even in resting macrophages, and is not due to its induction of marked inflammatory responses.
In order to investigate if the failure to grow in the mouse organs is due to the inability of the mutant to survive inside the macrophages, we examined the growth and survival of the mutant in resting and IFN-γ activated murine bone marrow derived macrophages which generate an oxidative burst by producing ROS and RNS [37].
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