After the flow cytometric or fluorometric readings were gathered, representative samples of cells were fixed in 3.5% paraformaldehyde solution for 20 min on ice, washed, and stained with the Hoechst 33342 dye (1 μg/ml), for nuclear staining. The slides were mounted with Vectashield (Vector Laboratories Inc., Burlingame, CA) and examined using confocal microscopy.
To confirm localization of bacteria within LAMP1-expressing phagolysosomes, representative fixed cells were permeabilized with 0.05% saponin solution and stained with Alexa Fluor 488-conjugated anti-mouse LAMP1 (expressed on lysosomal structures in cells) antibody (25 μg/ml; Biolegend, San Diego, CA).[36 (link)] Hoechst 33342 dye (1 mg/ml) was then added for nuclear staining. All images were acquired using a 63X oil immersion objective in a Zeiss confocal microscope, and were processed using the Zeiss ZEN 2011 program.
To confirm localization of bacteria within LAMP1-expressing phagolysosomes, representative fixed cells were permeabilized with 0.05% saponin solution and stained with Alexa Fluor 488-conjugated anti-mouse LAMP1 (expressed on lysosomal structures in cells) antibody (25 μg/ml; Biolegend, San Diego, CA).[36 (link)] Hoechst 33342 dye (1 mg/ml) was then added for nuclear staining. All images were acquired using a 63X oil immersion objective in a Zeiss confocal microscope, and were processed using the Zeiss ZEN 2011 program.
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