Visualization of Bacteria in Bladder Biopsy Sections via Fluorescence In Situ Hybridization

Abstract

Source: Neugent, M. L. et al., Detection of Tissue-resident Bacteria in Bladder Biopsies by 16S rRNA Fluorescence In Situ Hybridization. J. Vis. Exp. (2019)

This video demonstrates the visualization of bacteria in deparaffinized bladder tissue from a urinary tract-infected patient. Fluorescently-labeled probe hybridized with bacterial 16s ribosomal RNAs reveals the tissue-associated bacteria and their distribution, providing valuable spatial insights into their density within the sample.

Protocol

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.

1. Tissue Preparation for Fixation and Paraffin Embedding

NOTE: Biopsies were taken from consenting women undergoing cystoscopy with electro-fulguration of trigonitis for the advanced management of recurrent urinary tract infection (rUTI). rUTI is defined as 3 UTIs in 12 months. Biopsy collection was performed in the operating room while the patient was under anesthesia after obtaining informed patient consent. All samples were coded and de-identified before experi....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Alexa-555 Phalloidin	Invitrogen	A34055	Staining Actin
Alexa-555 Wheat Germ Agglutinin (WGA)	Invitrogen	W32464	Staining Mucin
Bottle top filters	Fisher Scientific	09-741-07	Sterilization
Coplin Jar	Simport	M900-12W	Deparaffinization/washing
Coverslips	Fisher Scientific	12-548-5M	Microscopy
Ethanol	Fisher Scientific	04-355-224	Rehydration
Ethylenediaminetetraacetic Acid, Disodium Salt Dihydrate	Fisher Scientific	S311-500	TE
Frosted Slides	Thermo Fisher Scientific	12-550-343
Hoechst 33342, Trihydrochloride, trihydrate	Invitrogen	H21492	Staining Nucleus
Hydrophobic marker	Vector Laboratories	H-4000	Hydrophobic barrier
Kimwipes	Fisher Scientific	06-666-11
Oil Immersol 518 F	Fisher Scientific	12-624-66A	Microscopy
Paraformaldehyde (16%)	Thermo Scientific	TJ274997	Fixation
ProLong Gold antifade reagent	Invitrogen	P36934	Mounting medium
Sodium Chloride	Fisher Scientific	BP358-10	Hybridization buffer/PBS
Sodium Dodecyl Sulphate	Fisher Scientific	BP166-500	Hybridization buffer
Sodium Phosphate Dibasic Hepahydrate	Fisher Scientific	S373-500	PBS
Sodium Phosphate Monobasic Monohydrate	Fisher Scientific	S369-500	PBS
Syringe	VWR	75486-756	Sterilization
Tris-HCl	Fisher Scientific	BP152-5	TE/Hybridization buffer
Xylene	Fisher Chemical	X3P-1GAL	Deparaffinization
0.22 micron syringe filter	Fisher Scientific	09-754-29	Sterilization