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 rotation.

At the end of the fixation, embed the organs in clear tissue freezing medium for storage at minus 80 degrees Celsius. Use a cryostat to acquire 10 micrometer thick tissue sections and dry the sections on individual pre-cleaned, charged glass slides for 20 minutes in the dark before applying hard mounting medium supplemented with DAPI. Then apply cover slips and cure the slides at room temperature overnight before transferring the samples to long-term storage at 4 degrees Celsius.

To identify lesions within the samples, place a slide on the stage of a laser scanning confocal microscope and use an appropriate objective for visualizing individual cells to acquire images of the lesions.