Nov 04, 2025

Public workspaceFluoresence microscopy shadow imaging of the neuropil and extracellular space in live organotypic mouse brain slices

DOI
https://dx.doi.org/10.17504/protocols.io.4r3l21zr3g1y/v1
  • Jan Tonnesen1
  • 1Instituto Biofisika of the Spanish National Research Council (CSIC)
  • Jan Tonnesen: Protocol developed in lab of Valentin Nagerl.
Jan Tonnesen
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DOI: https://dx.doi.org/10.17504/protocols.io.4r3l21zr3g1y/v1
Protocol Citation: Jan Tonnesen 2025. Fluoresence microscopy shadow imaging of the neuropil and extracellular space in live organotypic mouse brain slices. protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l21zr3g1y/v1
Manuscript citation:
Super-Resolution Imaging of the Extracellular Space in Living Brain Tissue
Jan Tønnesen , V.V.G. Krishna Inavalli, U Valentin Nagerl
Cell, 2018
https://doi.org/10.1016/j.cell.2018.02.007
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: November 03, 2025
Last Modified: November 04, 2025
Protocol Integer ID: 231419
Keywords: fluoresence microscopy shadow imaging, microscopy shadow imaging, extracellular space in live organotypic mouse brain slice, imaging in organotypic culture, live organotypic mouse brain slice, mouse brain, performing fluorescence, imaging, fluoresence, extracellular space, organotypic culture
Abstract
This protocol describes the key steps of performing fluorescence shadow imaging in organotypic cultures of mouse brain.
Materials
  • Scanning fluorescence microscope (e.g. confocal, 2-photon, or STED)
  • Calcein (Sigma, C0875)
  • NaCl (Sigma)
  • KCl (Sigma)
  • CaCl2 • 2H2O (Sigma)
  • MgCl2 • 6H2O (Sigma)
  • Glucose • H2O (Sigma)
  • HEPES (Sigma)
Troubleshooting
Shadow imaging protocol
Prepare a stock solution of 5 mM cacein fluorophore solution to add to the perfusion artificial cerebrospinal fluid (ACSF) . Calcein is dissolved in PBS and stored frozen at -20 degrees as 0.5 ml aliquots.
Prepare ACSF for perfusing the tissue in imaging chamber. Note this buffer does not require carbogen bubbling but can be used in ambient normal atmosphere.
ComponentConcentration (mM)
NaCl 126
KCl 2.5
CaCl2 • 2H2O 2.5
MgCl2 • 6H2O 1.3
Glucose • H2O 20
HEPES 28
Dissolve ingredients in MilliQ H2O, adjust pH to 7.3-7.4 with 1M NaOH if needed.
Prepare the given microscope you are using by warming up putative laser and turning on any temperature and atmosphere control. A laser scanning microscope is recommended as it offers better optical sectioning, e.g. a confocal, 2-photon, or STED microscope based on either of these modalities.
Transfer the organotypic culture to the microscope imaging chamber. For organotypic cultures grown on glass coverslips the tissue should always remain on the glass, also during imaging. For organotypic slices grown as membrane interface cultures, the slice is transferred on a small piece of membrane to the chamber.
Perfuse the slice with ACSF (typically 1-4 ml/min) and adjust the field of view to a given area of interest using brightfield video imaging and/or the microscope eyepieces.
Add calcein, Alexa Fluor 488, or other hydrophilic fluorophore to the ACSF at 40-50 μM final solution. It is recommended to not prepare more fluorophore contaning ACSF than necessary for a given experiment.
Let the fluorophore solution wash in and distribute homogeneoulsy in the tissue. This can be evaluated by time-lapse imaging of a set random area/volume and reading out when the fluorescence signal from the Calcein-ACSF reaches steady state. Usually this takes around 10 minutes.
Now everything is ready and imaging can start. As in any other fluorescent imaging experiment, it is recommended to optimize laser powers and detector gain for the given settings. Similarly, pixel size should be set taking into account the expected size of imaged structures and Nyquist's sampling theorem; e.g. 80 nm oixels for a good confocal microscope, or 200 nm pixels for a 2-photon microscope. Note that the fluoresence intensity of the ACSF can be adjusted on the fly by adding either fluorophore ir unlabelled ACSF to the calcein-ACSF solution.
After imaging has finished, the slice is discarded or fixed with PFA and saved for further processing, e.g. immunohistochemistry.
Protocol references
Super-Resolution Imaging of the Extracellular Space in Living Brain Tissue
Jan Tønnesen , V.V.G. Krishna Inavalli, U Valentin Nagerl
Cell 2018
https://doi.org/10.1016/j.cell.2018.02.007