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Acute isolation of hippocampal astrocytes protocol

Related

  • An overview of the functions of glia in the CNS
    • The role of glial cells in demyelinating diseases
      • Immunocytochemistry (ICC) and immunofluorescence (IF) protocol

        Procedure for isolating and visualizing hippocampal astrocytes from mice.

        Kindly submitted by Gerald Seifert and Christian Steinhäuser, Institute of Cellular Neurosciences, University of Bonn, Germany.

        Print this protocol.

        Procedure

        1. Dissect brains from postnatal day 5–30 mice and cut into 300 µm thick slices in frontal orientation by using a vibratome (HM 650V; Microm International, Walldorf, Germany).
        2. Perform slice preparation in an ice-cold, carbogen-saturated (95% O2/5% CO2) artificial cerebrospinal fluid (ACSF) containing sucrose (pH 7.4).
        3. Incubate for 30 min at 35°C in ACSF supplemented with sucrose, and store the slices for 20 min in ACSF. 
        4. Incubate the slices for 7–15 min in papain-containing (25 U/ml; Sigma P-4762 supplemented with L-cysteine monohydrate 0.8 mg/ml) ACSF at room temperature (bubbling with carbogen is necessary). For tissues from older mice incubate the slices for up to 15 min.
        5. After washing, dissect the CA1 region of the hippocampus and isolate cells in HEPES-buffered solution using wide-bore pipettes. The cell suspensions contain many astrocytes identified by their characteristic morphology, based on the fluorescence in the case of cell dissociation from hGFAP-EGFP or Cx43kiECFP mice, or after staining of astrocytes with SR101 (1 µM; Kafitz et al. 2008, J. Neurosci. Meth. 169:84–92).

        Perform SR101 incubation during ACSF/sucrose incubation at 35°C (Step 3).
        ​
        ​

        Reagents

        ACSF with sucrose

        87 mM NaCl
        2.5 mM KCl
        1.25 mM NaH​2PO4
        7 mM MgCl2
        0.5 mM CaCl2
        25 mM NaHCO3
        25 mM D-glucose
        75 mM sucrose gassed with carbogen

        ACSF without sucrose

        126 mM NaCl
        3 mM KCl
        1.25 mM NaH2PO4
        2 mM MgSO​4
        2 mM CaCl2
        26 mM NaHCO3
        10 mM D-glucose gassed with carbogen

        HEPES-buffered solution

        150 mM NaCl
        5 mM KCl
        2 mM MgSO​4
        2 mM CaCl2
        10 mM HEPES
        10 mM D-glucose gassed with O2
        Adjust solution pH to 7.4 using NaOH or HCl

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