Fluoroshield with 4 6 diamidino 2 phenylindole dapi

Human alveolar epithelial cells (A549) were seeded onto collagen-coated coverslips and incubated at 37°C and 5% CO₂ for 24 h. Cells were incubated in the presence of test agents for 2 h, after which the medium was replaced and the coverslips were incubated at 37°C and 5% CO₂ for 24 h. CellTracker red CMTPX dye (Thermo Fisher) was added to the cell medium for 30 min, the wells were washed with PBS, and green fluorescent protein (GFP)-expressing A. fumigatus conidia (a kind gift from William Hope, University of Liverpool) were added to wells to a final concentration of 1 × 10³ spores ml⁻¹. After 24 h of incubation at 35°C and 5% CO₂, coverslips were washed and affixed to slides by use of Fluoroshield with DAPI (4′,6-diamidino-2-phenylindole) (Sigma).

Nude mouse trypomastigotes derived from all strains were purified, counted, and incubated with HFF-1 and Swan 71 semiconfluent cell monolayers for 2 h at a multiplicity of infection of 5 parasites to 1 host cell. Afterwards, noninternalized trypomastigotes were washed 5 times with PBS and cells were incubated for another 48 h before immunofluorescence microscopy. After 48 h, coverslips were washed with PBS, fixed with 95% (vol/vol) ethanol, and stained with Fluoroshield with DAPI (4′,6-diamidino-2-phenylindole; Sigma, USA). Infectivity was evaluated considering invasion and replication capacity by counting infected cells and parasites per infected cell in the infection photos obtained from each experimental condition. For each replicate, a total of at least 500 cells were counted and the results are expressed in graphs as the mean values and standard errors (SE) of 3 independent experiments. The infection index was calculated as follows: infection index = (number of amastigotes × number of infected cells)/total number of cells.

Formalin-fixed and paraffin-embedded tissue sections of spleen were mounted on poly-l-Lysine pre-coated slides (Sigma, USA) followed by deparaffinization in xylene, rehydration in graded ethanol, and quenching of endoperoxidase activity using 3 % H₂O₂ in methanol. Antigen retrieval was done in heat-mediated antigen retrieval solution according to the manufacturer’s instructions (Vector labs, USA). Then, 2.5 % normal non-immune goat serum (Vector labs) was used to block non-specific sites for 1 h at room temperature. For, both IHC and IFT, sections were incubated with rabbit anti-brucella hyper-immune serum (1 : 20) (supplied from the Division of Biological products, ICAR-IVRI) as primary antibodies. For IHC, sections were incubated with goat anti-rabbit IgG (whole molecule)-peroxidase conjugate (1 : 200) (Sigma, USA) and developed using a DAB (3,3′-diaminobenzidine tetrahydrochloride) enhanced liquid-substrate system (Sigma, USA). Immunostained sections were counter stained with Mayer’s hematoxylin (Sigma, USA) and mounted with tissue mounting medium (CC/ Mount, Sigma, USA). For IFT, goat anti-rabbit IgG (whole molecule) – FITC conjugated secondary antibodies (Sigma, USA) was used (1 : 40) followed by mounting using Fluoroshield with DAPI (4′,6-diamidino-2-phenylindole) (Sigma, USA) and viewed under fluorescent microscope (Nikon Eclipse Ti-S, Japan).