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 …
Açıklamalar
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 |
Bu Makaleden Alıntı Yapın
Visualization of Bacteria in Bladder Biopsy Sections via Fluorescence In Situ Hybridization. J. Vis. Exp. (Pending Publication), e21837, doi: (2023).