Preparation of Mouse Bladder Tissue for Visualization of Bacterial Infection

Begin with a euthanized mouse with an exposed bladder, pre-infected with pathogenic bacteria.

The bacteria invade the urothelium and form colonies. Upon maturation, these bacteria rupture the epithelial cell and infect the surrounding cells.

Insert a catheter connected to a syringe filled with fixative. Slowly instill the fixative to inflate the bladder and preserve its architecture.

Clamp the urethra to retain the fixative and maintain bladder shape.

Remove the catheter, then cut the urethra. Transfer the bladder to fresh fixative and incubate to preserve the structure.

Section the bladder into two half-bladder cups to expose the urothelium, and rinse with a buffer.

Stain with a metal-based reagent to enhance contrast, then wash with ultrapure water.

Dehydrate the sections using an ethanol gradient, followed by critical-point drying.

Cut each dried bladder cup in half.

The sample is now ready for downstream processing to visualize host-pathogen interactions.

Once tuberculin slip-tip syringe with fixative, affix a catheter to the end, bevel facing opposite the syringe markings.

Snip off the excess tubing 1 to 2 millimeters from the end of the needle, taking care not to expose the needle tip. Flick the syringe to remove bubbles and push the plunger to void air. Then fill the catheter with fixative over a microcentrifuge tube. After anesthetizing and sacrificing the mouse, once the legs are secured, open the mouse's pelvic area with forceps and a pair of surgical scissors to expose the bladder. Carefully push aside the adjacent fat, but leave the bladder in place.

Hold the syringe with the dominant hand with the needle pointing down. Dip the catheter tip into sterile lubricant and position the catheter tip at the urethral opening, holding the syringe barrel away at a 30 to 45 degree angle over the mouse body.

Apply downward pressure with slight clockwise motion and gently insert the catheter into the urethra. As the catheter tip enters, hinge the syringe toward the tail of the mouse while continuing to slide the catheter further into the urethra until the syringe barrel is parallel to the working surface. The entire catheter needle shaft should enter the mouse, positioning the catheter tip within the bladder lumen. Slowly deliver 50 to 80 microliters of fixative, causing the bladder to inflate like a balloon.

Keep the catheter in place and raise the syringe slightly, tilting the tip up. With the other hand, open a hemostat and slide one prong under the catheter needle at the intersection of the urethra. Partially close the hemostat until it just makes contact with the needle.

Gently slide the catheter needle out of the bladder while simultaneously clamping down and locking the hemostat completely to prevent loss of the fixative. Grip the hemostat so that it is parallel to the working surface with the bladder resting on top. Lift up gently and carefully cut under the hemostat to remove the bladder with the hemostat still attached.

Place the bladder and attached hemostat into a Falcon tube containing warmed fixative. Ensure that the bladder is fully submerged in the fluid and not pressed against the walls of the tube.

In order to image the bladder by scanning electron microscopy, sagittally bisect it with a clean, double-sided razor blade or scalpel, and make a second cut tangential to the hemostat to release the bladder. This results in two half-bladder cups. If any remaining fat pads exist on the exterior of the bladder, gently remove them.

Rinse the bladder halves three times in sodium cacodylate buffer. Stain the tissue with 1% osmium tetroxide in 0.15 molar cacodylate buffer for 1 hour at room temperature. Perform this step with the staining vessel wrapped in foil to maintain a dark environment. After staining, rinse the bladder halves three times in ultra-pure water.

If osmicated oil is seen on the surface of the water, aspirate or wick it off to prevent contamination during the drying steps. After dehydrating the tissue, bisect the bladder halves with a clean, double-sided razor to generate four total pieces.