Start with an immobilized, dissected mouse bladder pre-infected with β-galactosidase-expressing uropathogenic E. coli, placed in a silicone-coated multi-well plate.
The bladder’s lumen faces up, exposing the epithelial cells that contain intracellular bacterial communities, or IBCs.
Wash the bladder with a buffer to remove debris and unattached bacteria.
Next, add a fixative and incubate to preserve cellular and bacterial structures.
Remove the fixative and wash with a buffer.
Then, wash with a detergent-containing buffer to permeabilize cellular and bacterial membranes.
Remove the buffer, then add a solution of X-gal, a substrate of β-galactosidase, supplemented with redox reagents, and incubate.
X-gal and redox reagents enter the bacteria. β-galactosidase cleaves X-gal, producing a colorless intermediate that is oxidised by redox reagents to form a blue precipitate.
Under a dissecting microscope, the blue dots in the bladder confirm the IBCs, indicating a urinary tract infection.
For IBC enumeration, after harvesting the bladder, place it in a silicone-coated well of a 6-well plate with PBS and use scissors to cut it in half.
Using small metal pins placed along the outermost edges, gently splay the bladder so that the lumen is facing upward and the maximum amount of urothelium is exposed. After performing LacZ stain as outlined in the text protocol, observe IBCs, which appear as blue puncta under a dissecting microscope.
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