This article details a protocol for visualizing Streptococcus pneumoniae in cardiac tissue using immunofluorescence microscopy. The method involves several steps including fixation, permeabilization, blocking, and antibody staining to highlight bacterial presence within microlesions.
Begin with a slide containing a chemically fixed cardiac tissue section from a mouse infected with Streptococcus pneumoniae, a pathogenic bacterium.
This infection causes extracellular, bacteria-filled microlesions to form within the cardiac muscles.
Wash the section with a buffer to remove any residual fixative.
Apply a non-ionic detergent to permeabilize the host cellular membranes.
Wash again to eliminate any remaining detergent.
Incubate the section with a blocking agent to block nonspecific binding sites.
Rinse to remove unbound blocking agent.
Introduce a primary antibody that binds to the capsular polysaccharide of the bacteria.
Wash to remove any unbound antibody, then add a green fluorophore-labeled secondary antibody that binds to the primary antibody.
Counterstain with a nuclear dye to label host cell nuclei.
Wash the section and mount it using a mounting medium.
Observe the section using confocal microscopy to visualize labeled bacteria within the microlesions.
Using a 5-micron cardiac section on positively charged glass slides, first freeze the sections, then thaw them and allow them to air dry. Fix the slides in 10% neutral buffered formalin for 10 minutes at 25 degrees Celsius. Then, remove the fixative with three washes and PBS for five minutes per wash.
Follow by permeabilizing the tissue with 0.2% Triton X-100 in PBS for 15 minutes. Remove the detergent with three more washes in straight PBS. Then, block the tissue with 10% goat serum in PBS for an hour. After a rinse in PBS, cover the section with antiserum for two hours at 37 degrees Celsius. Treat negative controls with naive rabbit antiserum.
Because cardiac tissue readily absorbs the XI Immunofluorescent stain, leading to high background levels, the concentration in incubation time for different types of antibodies will likely need to be optimized.
Two hours later, wash the anti-serum away with PBS. Then replace the solution with a secondary antibody and let the slides incubate at 37 degrees Celsius for another 30 minutes. Now, apply DAPI at 5 milligrams per milliliter and wash the slides with PBS. Finally, mount the slides in FluorSave and image them with confocal microscopy.
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