After fixation, tissue slides were embedded in paraffin. Four to 5 μm-thick sections were cut and stained with hematoxylin and eosin (HE). These sections were examined by at least two of the authors who were blind to previous knowledge of the dogs’ health and identities.
The authors scored the level of white pulp organization: (1) slightly disorganized, with either hyperplastic or hypoplastic changes leading to a loss of definition of any of the regions of the white pulp and (2) for moderately or extensively disorganized, when the white pulp regions were poorly individualized or indistinct. IHC stains were performed using the standard streptavidin–biotin peroxidase (HRP) immunostaining procedure with polyclonal antibody.
Anti-CD3 (A0452) (DAKO, CA, USA) was used to detect T lymphocytes. This antibody was previously used to stain CD3 in canine lymphomas (Sueiro et al., 2004). Slides were deparaffinized and hydrated. Antigen retrieval JQ1 was achieved by steam heating in citrate buffer for 30 min. For inhibition of endogenous peroxidase, slides were incubated with 2% (v/v) hydrogen peroxide 30 vol diluted in 50% (v/v) methanol for 30 min and nonspecific binding was blocked with 3% (w/v) nonfat dry milk in PBS for 30 min. Primary antibody against CD3 (1:100) was applied for 18–22 h at 4 °C in a humidified chamber. Slides were washed in PBS, incubated with a biotinylated secondary antibody (LSAB+ Kit, DAKO K0690, CA, USA) for 45 min at room temperature, washed once
more with PBS, and incubated with streptavidin–HRP complex (LSAB+ Kit, DAKO selleck inhibitor K0690, CA, USA) for 45 min at room temperature. The reaction was developed with 3,3′-diaminobenzidine (DAKO K3468, CA, USA). The slides were counterstained with Harris’s hematoxylin, dehydrated, cleared and mounted with coverslips. Spleen tissue from healthy dogs was used as a positive control. The data were analyzed by a nonparametric test. Group means were compared using Mann Whitney test. The least squares method was used to evaluate the effect of group (degree of correlation for the structural Rebamipide organization of white pulp) and quantitative variable (percentage of apoptosis). The results were considered significant when P < 0.05. The SAS software was used (SAS 9.1, SAS Institute, Cary, NC, USA) for all statistical analyses performed in this study. Flow cytometry analysis of CD3 lymphocytes in PMBC from infected dogs showed significantly lower numbers (58 ± 12, mean ± SD) compared to healthy controls (80.6 ± 5, mean ± SD) (Fig. 1) (P = 0.001, Mann Whitney test). To examine apoptosis in T cells from PBMC and spleen of L. chagasi-infected dogs, PBMC and spleen samples were evaluated immediately following collection. Apoptosis of T cells from PBMC and spleen was detected using commercial kits for both, Annexin V (Guava, Hayward, CA) and Caspases (Guava, Hayward, CA) and simultaneously anti-CD3 mAbs (Serotec, UK).