To visualize cell nuclei and intracellular HIV protein, paraformaldehyde-fixed MDM cells were treated with 0.1% Igepal and 1% saponin for 20 min, washed with 1× PBS and incubated for 1 hour with human sera as a blocking agent. Cells were then incubated with (500 ng/ml) of DAPI in 0.1% Igepal for 10 min and washed twice with 1× PBS. Anti-p24 monoclonal antibody (Thermo Scientific, MA1–7040) was conjugated to Alexa-Fluor 647 (Invitrogen) as per manufacturer’s instructions and purified by column chromatography (GE Healthcare) followed by desalting columns (Thermo Scientific). MDM were stained with 0.1 μg/ml monoclonal antibody for 1 hour, washed with 1× PBS three times and coated with 50% glycerol + n-propyl gallate (5 mg/ml) for imaging.
To visualize iNOS expression in Mtb-infected macrophages, macrophages were fixed for 2 h with 2% PFA at room temperature (RT). After fixation, the samples were blocked in 0.3% Triton-X 100/5 % donkey serum serum/1X PBS for 60 min at RT, washed, and incubated with anti-NOS2 (Cell Signaling Technology Clone D6B6; 1:100) overnight at 4 °C. The samples were washed again and stained with an Alexa Fluor 647–conjugated donkey anti-rabbit secondary antibody (Thermo Fisher; 1:1000, A-31573) for 1 h at RT. Images were obtained using a Zeiss LSM 510 Confocal Microscope fitted with a 20X objective.
To visualize iNOS expression in Mtb-infected macrophages, macrophages were fixed for 2 h with 2% PFA at room temperature (RT). After fixation, the samples were blocked in 0.3% Triton-X 100/5 % donkey serum serum/1X PBS for 60 min at RT, washed, and incubated with anti-NOS2 (Cell Signaling Technology Clone D6B6; 1:100) overnight at 4 °C. The samples were washed again and stained with an Alexa Fluor 647–conjugated donkey anti-rabbit secondary antibody (Thermo Fisher; 1:1000, A-31573) for 1 h at RT. Images were obtained using a Zeiss LSM 510 Confocal Microscope fitted with a 20X objective.