Fluorescence Imaging of Bacterial Colonization in Mouse Kidney Tissue

Begin with a mouse kidney infected with engineered Gram-positive bacteria that colonize the tissue and form lesions.

All bacterial cells express red fluorescence, while lesion-specific host conditions enable green fluorescent protein expression in specific cells.

Treat the kidney with a fixative and incubate in the dark to preserve the tissue structure and partially permeabilize cell membranes.

Embed the organ in a suitable medium, freeze it, and section it into thin tissue slices.

Place the slice onto a charged glass slide for adhesion.

Stain the tissue with a nuclear dye.

The dye penetrates host and bacterial cells and binds to DNA.

Place a coverslip over the tissue and observe the slide under a fluorescence microscope using specific imaging channels.

Nuclear staining outlines host and bacterial DNA.

All bacteria in the tissue express red fluorescence, while green fluorescence expression indicates the site of host-pathogen interaction.

At the appropriate experimental endpoint, harvest the kidney, heart, liver, lungs, and spleen into 15 milliliter polypropylene tubes containing 10% buffered formalin for a 24 to 48 hour incubation in the dark at room temperature with gentle shaking or rotati