ProtocolA fluorescence confocal assay to assess neuronal viability in brain slices
Section snippets
Type of research
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Ischemia and neurodegeneration.
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Neuroprotection and neuronal viability.
Time required
The time required is dependent upon the number of slices to be assayed. The time to run the complete neuroprotection assay in hippocampal slices is 7–10 h; however, the viability assay itself requires only 2 h to assay six slices:
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Isolation of the hippocampus from the rat brain requires 5 min.
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Slice preparation requires 10 min/hippocampus.
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Recovery time for slices prior to treatment is 90 min.
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Slices are pre-incubated with the test compounds for 30 min.
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The hypoxic-hypoglycemic insult is 10 min in
Materials
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An inverted confocal laser scanning microscope equipped to detect green and red emitted light. We use a Zeiss LSM 410 (Carl Zeiss, Thornwood, NY) microscope equipped with simultaneous recording on 3 PMTs and an Omnichrome krypton-argon laser (Chino, CA).
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20× and 5× infinity corrected fluorescence objectives. We use Zeiss Achroplan 20× (0.4 NA) and Zeiss Fluar 5× (0.25 NA) objectives.
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Dichroic beam splitters: beam splitter 1: DBSP 488/568; beam splitter 2: FT560.
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Excitation line selection filter:
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Preparation of hippocampal slices
All animal procedures have been approved by the Institute for Biological Sciences Animal Care Committee. Male Wistar rats (180–230 g) receive Purina rat chow and water ad libitum. Anesthetize rats with 3% halothane, decapitate and remove their brains rapidly (<1 min) and submerge them in cold (4°C) ACSF consisting of (in mM): 127 NaCl, 2 KCl, 10 glucose, 1.2 KH2PO4, 26 NaHCO3, 2 MgSO4, 2 CaCl2 (pH 7.4) bubbled with 95% O2/5% CO2. Dissect both hippocampi and cut 400 μm transverse slices with a
Results
In the CA1 region of control slices and oxygen-glucose deprived slices at 60 and 120 min of reperfusion there was an abundance of live, green cells and few dead, red cells (Fig. 3A–C; [38]). By comparison, oxygen-glucose deprived slices following 240 min of reperfusion possessed mainly dead, red and few live, green cells (Fig. 3D; [38]). Occasionally, with severe insults cell bodies were visibly absent.
When slices which were to be deprived of oxygen and glucose were incubated in a low (0.3 mM)
Slice staining procedure
Calcein-AM is a non-fluorescent membrane permeable esterase substrate that is passively loaded into cells 5, 17, 31. When the acetoxymethyl ester portion of the molecule is hydrolyzed, the released product, calcein, which is membrane impermeable, is trapped in the cytoplasm of cells with intact membranes. Calcein emits a green (517 nm) fluorescence under blue light excitation (497 nm). Thus, the green fluorescence of calcein indicates cells that have an intact membrane and esterase activity
Preparation of hippocampal slices
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Extract rat brain, dissect hippocampus and cut 400 μm transverse slices.
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Submerge slices in 2–3 mm of ACSF bubbled with 95% O2/5% CO2 for 90 min at 36.5°C.
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Pretreat slices for 30 min with test compound.
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Put slices to be made ischemic in insult chamber for the 10 min hypoxic-hypoglycemic insult.
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Reperfuse for 4–6 h.
Confocal microscopy
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Stain slices with 4 μM calcein and 6 μM ethidium homodimer for 30 min in ACSF.
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Wash slices in ACSF for at least 15 min.
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Determine confocal sensitivity parameters by imaging control slices
Essential literature references
Refs. 4, 15, 17, 22, 30, 35, 40.
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