Preparation of Mouse Bladder Tissue for Visualization of Bacterial Infection

Abstract

Source: O'Brien, V. P., et. al., Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice. J. Vis. Exp. (2020)

This video demonstrates the use of scanning electron microscopy to visualize bacterial colonization and host–pathogen interactions in the bladder epithelium of a pre-infected mouse model.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Imaging bladders by scanning electron microscopy

   NOTE: This procedure can be performed at any time point at which visualization of the urothelium is desired. As indicated in Figure 1 (purple boxes), uropathogenic E. coli (UPEC) -urothelial interactions are best visualized between 6 h and 24 h post-UPEC inoculation during the reservoir formation phase, and urothelial exfoliation triggered by Gardnerella vaginalis is best visualized between 3 h and 12 h after the s....

Results

Figure 1. Schematic of Mouse Model. The timeline is highlighted to reflect the phases or procedures of the model outlined in the protocol. Phase 1 (orange): Establishing intracellular uropathogenic E. coli (UPEC) reservoirs. Mice are .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
30G x 1/2 needles	BD	305106	For catheters
5 1/2" straight forcep hemostat	McKesson	487377	In situ bladder fixation
ACE 600 Sputter coater	Leica		SEM sample processing
Aluminum SEM stub	Ted Pella	16111	SEM sample processing
Calcium chloride	EMS	12340	In situ bladder fixation
Conductive carbon adhesive tab	Ted Pella	16084-1	SEM sample processing
Conductive silver paint	Ted Pella	16034	SEM sample processing
CPD 300 Critical Point Drier	Leica		SEM sample processing
Cytofunnel metal clip	Simport	M964B	cytospun urinalysis
Ethanol	EMS	15050	SEM sample processing
Glucose	Sigma	G7528	For NYCIII G. vaginalis growth media
Glutaraldehyde	EMS	16320	In situ bladder fixation
Hema 3 staining kit	Fisher	23123869	Cytospun urinalysis
HEPES	Cellgro	25-060-Cl	For NYCIII G. vaginalis growth media
Iridium	Ted Pella	91120	SEM sample processing
Merlin FE-SEM	Zeiss		Scanning electron microscope
Milli-Q Water Purifier	Millipore	IQ-7000	SEM sample processing
Osmium tetroxide	EMS	19170	SEM sample processing
Paraformaldehyde	EMS	15710	In situ bladder fixation
Polyethylene tubing	Intramedic	427401	for catheters
Proteose Peptone #3	Fisher	DF-122-17-4	For NYCIII G. vaginalis growth media
PTFE coated double edge razor blade	EMS	72000	Cutting bladders for SEM