Dmre fluorescence microscope

5 x 10³ MEFs were grown on glass coverslips in multiwell plates and infected with 10 moi of HSV-1 or SFV. At 18h post-infection, the cells were fixed in 4% paraformaldehyde and permeabilized in 0.1% Triton X-100 (Sigma). The cells were then incubated with mouse monoclonal anti-gD or mouse monoclonal anti-cytochrome c and rabbit polyclonal anti-caspase-3 antibodies followed by FITC-conjugated goat anti-mouse IgG, (Millipore) and PE-conjugated goat anti-rabbit IgG, F(ab’)₂ fragment (Santa Cruz Biotechnology) secondary antibodies (1:200) for 90 min. Nuclei were stained with Hoechst 33334 (5 mg/ml; Sigma). The samples were directly viewed under a Leica DMRE fluorescence microscope.

Cardiomyocytes were dissociated from cell culture plates as described in “Generation of hiPSC-CMs”. The separated cells were resuspended with serum-free medium and transferred to 4 chamber glass slides (Corning). After resting for 48 h the cells were washed with PBS. Fixation was performed using 4% paraformaldehyde (Roti-Histofix Roth cat# P087.4) for 10 minutes. Afterwards the cells were permeabilized for 10 minutes with 0,5% Triton X-100 (Sigma cat# T-9284) and blocked with PBS containing 1% bovine serum albumin (Sigma cat# A-6003). After each step they were washed with PBS. Finally cells were incubated with several antibodies at 4 °C in the dark. After one hour antibodies were washed out with PBS. Nuclear staining was performed with Vectashield mounting medium with DAPI (Linaris cat# H-1200). The antibodies used in this study were FITC-conjugated cardiac troponin T antibody (1:66 dilution, biorbyt cat# orb187249), cy5-conjugated titin antibody (1:66 dilution, BiossInc cat# bs-9861R-Cy5), Alexa Fluor 488-conjugated NfκB-p65 subunit antibody (1:40 dilution, BD biosciences cat# 558421). Pictures were taken with Leica DMRE fluorescence microscope, DFC3000G/DFC 450c camera equipped with Leica Application suite 4.4 software.

MPPa sparkles with red fluorescence upon corresponding excitation. To observe the uptake of MPPa in MCF-7 cells, cells were seeded into 6-well plates and incubated with 2 μmol/L MPPa for different times (0 h, 3 h, 6 h, 12 h, and 24 h). The intracellular accumulation of MPPa was detected by fluorescence microscopy (Leica DMRE Fluorescence Microscope, Germany).

To examine passage of trypanosomes across the BBB, 14 μm vertical sections of fresh frozen brains at levels including the septal nuclei were cut, mounted and fixed in 4% formalin with 0.17% picric acid in PBS followed by acetone. The sections were double labeled by incubation with either antibodies recognizing the parasite, cerebral endothelial and inflammatory cells or plasma proteins (S2 Table), followed by fluorochrome labelled secondary antibodies rhodamine red donkey anti-rat IgG, anti-rabbit IgG, or anti mouse IgG and Alexa Fluor488 donkey anti-goat IgG (all from Jackson Immunoresearch, West Grove, PA). Sections were examined in a Leica DMRE fluorescence microscope.

Fixation and preparation of bacterial cells for immunofluorescence microscopy was performed as previously (Browning et al., 2013 (link)). Briefly, fixed cells were put onto poly-L-lysine-coated coverslips, washed three times with PBS and then blocked for 1 h in PBS containing 1% [v/v] bovine serum albumin (Europa Bioproducts). Coverslips were incubated with 1:500 primary antibody for 1 h, washed three times with PBS, and incubated for an additional 1 h with Alexa Fluor^® 488 goat anti-rabbit IgG. The coverslips were washed a further three times with sterile PBS before mounting onto glass slides and visualised using either phase contrast or fluorescence using Leica DMRE fluorescence microscope (100 × objective) -DC200 digital camera system.

To collect intracellular bacteria at each infection time point, infected cells were washed twice with 1× PBS and lysed by adding 1% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA)) for 5 min. To evaluate the percentage of intracellular dead bacteria, recovered bacteria were pelleted, washed with 1× PBS and suspended in 1× PBS containing 10 μg/mL DAPI to stain the entire population and 15 μM Propidium Iodide (PI) to label dead bacteria. After 20 min of incubation at room temperature in the dark, bacteria were washed with 1× PBS and resuspended in 5 μL 1× PBS containing 50% glycerol. The entire sample was spotted on the glass slide and overlaid with the coverslip for immediate observation and counting under the fluorescence microscopy. Samples were examined using a Leica DMRE fluorescence microscope equipped with a 100× lens.
To define the number of viable intracellular bacteria, infection of epithelial cells was carried out as describe above. At the indicated time points, the cell lysate containing intracellular bacteria was collected, washed and resuspended in 1× PBS. Serial dilutions of the bacterial suspensions were plated on LB agar plates to calculate the CFU/mL.