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

Published: November 30, 2023

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 …

Disclosures

The authors have nothing to disclose.

Materials

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

Play Video

Cite This Article
Visualization of Bacteria in Bladder Biopsy Sections via Fluorescence In Situ Hybridization. J. Vis. Exp. (Pending Publication), e21837, doi: (2023).

View Video