Fluorescence Imaging of Bacterial Colonization in Mouse Kidney Tissue

Samenvatting

Source: Behera, R. K., et al. A Fluorescence-based Method to Study Bacterial Gene Regulation in Infected Tissues. J. Vis. Exp. (2019)

This video demonstrates a method for preparing infected mouse kidney tissue, staining DNA, and detecting bacterial gene expression using dual fluorescent reporters. Red marks all bacteria, and green highlights infection-responsive cells.

Protocol

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.

1. Harvesting the organs and tissue processing
   1. Euthanize the animals by CO₂ inhalation and cervical dislocation as a secondary method, and perform necropsy.
   2. Harvest the kidney (right) and other vital organs (heart, liver, lungs, spleen) and transfer into 15 mL polypropylene tubes containing 10% [v/v] buffered formalin.
   3. Allow the organs to fix in the dark at room temperature with gentle shaking or rotation for at least 24 hours but no more than 48 hours.
   4. Embed organs in a clear tissue freezing medium and store the tissues at -80 °C.
   5. Using a cryostat, section tissue into slices of 10 µm thickness.
   6. Dry the sections on a pre-cleaned, charged glass slide for 20 min in the dark. Apply a hard mounting medium with 4',6-diamidino-2-phenylindole (DAPI) stain and a coverslip. Cure mounted slides at room temperature overnight and transfer to 4 °C for long-term storage.
2. Laser Scanning Confocal Microscopy and Image Processing
   1. Examine mounted slides to locate lesions using lasers appropriate for the fluorescent reporter being used. In this case, the green (GFP or green fluorescent protein), red (tdTomato), and blue (DAPI) fluorescence signals are used.
   2. Acquire the image using the appropriate objective for visualizing individual cells (e.g., typically 20x or 40x objectives are used).

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
C57Bl/6 Mice	Charles River	NA
Confocal laser scanning microscope	Zeiss	NA
Cryostat microtome	Thermo Scientific	NA
Phosphate buffered saline (PBS)	Cellgrow	46-013-CM
Precellys 24 homogenizer	Bertin Laboratories	NA
Sub Xero clear tissue freezing medium	Mercedes Medical	MER5000
Superfrost Plus microscope slides	Fisher Scientific	12-550-15
Vectashield Antifade Mounting medium with DAPI	Vector Laboratories	H-1500