Immunostaining of Bacteria-Filled Microlesions in a Mouse Cardiac Tissue Section for Imaging

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.