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Biocytin Staining of Glia and Neurons in Brain Slices Jian Kang Cold Spring Harb Protoc; doi: 10.1101/pdb.prot083527 Email Alerting Service Subject Categories
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Protocol
Biocytin Staining of Glia and Neurons in Brain Slices Jian Kang
This protocol describes the use of biocytin to visualize and distinguish the morphology of glia and neurons in rat brain slices. Patch pipettes are used to load biocytin into different cell types. The slices are subsequently fixed, stained, and mounted in preparation for imaging.
MATERIALS It is essential that you consult the appropriate Material Safety Data Sheets and your institution’s Environmental Health and Safety Office for proper handling of equipment and hazardous materials used in this protocol. RECIPES: Please see the end of this protocol for recipes indicated by . Additional recipes can be found online at http://cshprotocols.cshlp.org/site/recipes.
Reagents
Albumin Biocytin hydrochloride (Sigma-Aldrich) Brain slices from 8- to 20-d-old (PND 8–20) Sprague–Dawley rats of either sex Fixative for biocytin staining Iodine Permount (Fisher Scientific) Phosphate-buffered saline (PBS) (0.1 M; Mediatech, Inc.) Pipette solution for biocytin staining Slice solution Triton X-100 Vectastain Elite ABC kit (Vector Labs) Equipment
Patch pipettes and other electrophysiology equipment Recording chamber (custom made) METHOD
1. Place the brain slices in a recording chamber (1.5 mL) and perfuse with slice solution. 2. Using two patch pipettes, load freshly prepared biocytin solution (pipette solution containing 5% biocytin hydrochloride) into each cell under study. For example, patch an interneuron with Adapted from Imaging in Neuroscience (ed. Helmchen and Konnerth). CSHL Press, Cold Spring Harbor, NY, USA, 2011. © 2014 Cold Spring Harbor Laboratory Press Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot083527
948
Downloaded from http://cshprotocols.cshlp.org/ at TULANE UNIV on September 8, 2014 - Published by Cold Spring Harbor Laboratory Press
Biocytin Staining
a biocytin-containing pipette, and then patch an astrocyte with another biocytin-containing pipette. 3. Without moving the patch pipettes, perfuse the slices with fixative for 10 min and then carefully withdraw the pipettes. 4. Postfix the slices for 10–12 h. Wash four times in 0.1 M PBS. 5. Incubate the slices in 0.1 M PBS containing 0.3% iodine at room temperature for 15 min. Wash 4× in PBS. 6. Incubate the slices in 0.1 M PBS containing 0.2% Triton X-100 and 0.2% albumin for 45 min at room temperature. Wash 4× in PBS. 7. Stain the fixed slices with the Vectastain Elite ABC kit. Mount with Permount.
DISCUSSION
We have used biocytin staining to confirm the identification of neural cells revealed by differential interference contrast (DIC) microscopy. Figure 1A illustrates a characteristic field of the stratum radiatum, containing both astrocytes and neurons, under DIC optics. Interneurons are characterized by large and bright somata (diameter 16.1 ± 2.7 µm; mean ± SD) with large dendrites, whereas astrocytic somata are small and round (diameter 8.1 ± 1.7 µm; mean ± SD) without large processes. The cell identified by DIC as an astrocyte was shown by biocytin staining (Fig. 1B) to have an extensive array of branched short processes, which are phenotypical for astrocytes. Nearby astrocytes could also be stained through gap junctions (Robinson et al. 1993). In contrast, the presumed interneuron was shown to have few evenly sized and less branched processes, a morphology that is stereotypical for neurons. The size of the biocytin-stained astrocyte seems larger than that of the same cell viewed under DIC optics. This might result from the mixture of short processes with the cell body. In this
FIGURE 1. Identification of interneurons and astrocytes in hippocampal slices. An astrocyte (white arrow; characterized by a small, rounded cell body and poorly defined, irregular processes) and an interneuron (red arrow; characterized by a large soma with large dendrites that project in all directions) located in the stratum radiatum were patched by two pipettes. After visualization under DIC optics (A), biocytin (0.5%) was injected into both cells to reveal cellular morphology (B). Scale bar, 10 µm. Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot083527
949
Downloaded from http://cshprotocols.cshlp.org/ at TULANE UNIV on September 8, 2014 - Published by Cold Spring Harbor Laboratory Press
J. Kang
experiment, a total of eight pairs of interneurons and astrocytes were injected with biocytin, and in all cases, biocytin staining confirmed cell identification under DIC.
RECIPES Fixative for Biocytin Staining
Add 40 mL of saturated picric acid (Sigma-Aldrich) and 80 mL of 25% glutaraldehyde (Fisher Scientific) to 9.88 mL of 0.2 M phosphate-buffered saline (Sigma-Aldrich). Dilute this solution 1:1 with 8% paraformaldehyde solution. Adjust the pH to 7.4 with NaOH. The final concentrations are 4% paraformaldehyde, 0.2% picric acid, and 0.1% glutaraldehyde. Paraformaldehyde Solution (8%)
Add 40 g of paraformaldehyde (Sigma-Aldrich) to 400 mL of distilled water. Heat to 60˚C and add 2–3 drops of 1 N NaOH. Adjust the final volume to 500 mL with distilled water. Store this stock solution at 4˚C. Pipette Solution for Biocytin Staining
Reagent Potassium gluconate KCl MgCl2 HEPES EGTA CaCl2 ATP Guanosine triphosphate (GTP) Glucose
Final concentration 120 mM 10 mM 1 mM 10 mM 0.1 mM 0.01 mM 1 mM 0.2 mM 4 mM
Adjust the pH to 7.2 with KOH. Slice Solution
Reagent NaCl KCl NaH2PO4 MgCl2 CaCl2 Glucose NaH2CO3
Final concentration 126 mM 2.5 mM 1.25 mM 2 mM 2 mM 10 mM 26 mM
The pH should be 7.4 when the solution is gassed with 5% CO2 and 95% O2 at room temperature (23˚C–24˚C).
REFERENCES Robinson SR, Hampson EC, Munro MN, Vaney DI. 1993. Unidirectional coupling of gap junctions between neuroglia. Science 262: 1072–1074.
950
Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot083527
Biocytin Staining of Glia and Neurons in Brain Slices Jian Kang Cold Spring Harb Protoc; doi: 10.1101/pdb.prot083527 Email Alerting Service Subject Categories
Receive free email alerts when new articles cite this article - click here. Browse articles on similar topics from Cold Spring Harbor Protocols. Cell Imaging (463 articles) Imaging for Neuroscience (274 articles) Labeling for Imaging (301 articles) Patch Clamping (40 articles)
To subscribe to Cold Spring Harbor Protocols go to:
http://cshprotocols.cshlp.org/subscriptions
© 2014 Cold Spring Harbor Laboratory Press
Downloaded from http://cshprotocols.cshlp.org/ at TULANE UNIV on September 8, 2014 - Published by Cold Spring Harbor Laboratory Press
Protocol
Biocytin Staining of Glia and Neurons in Brain Slices Jian Kang
This protocol describes the use of biocytin to visualize and distinguish the morphology of glia and neurons in rat brain slices. Patch pipettes are used to load biocytin into different cell types. The slices are subsequently fixed, stained, and mounted in preparation for imaging.
MATERIALS It is essential that you consult the appropriate Material Safety Data Sheets and your institution’s Environmental Health and Safety Office for proper handling of equipment and hazardous materials used in this protocol. RECIPES: Please see the end of this protocol for recipes indicated by . Additional recipes can be found online at http://cshprotocols.cshlp.org/site/recipes.
Reagents
Albumin Biocytin hydrochloride (Sigma-Aldrich) Brain slices from 8- to 20-d-old (PND 8–20) Sprague–Dawley rats of either sex Fixative for biocytin staining Iodine Permount (Fisher Scientific) Phosphate-buffered saline (PBS) (0.1 M; Mediatech, Inc.) Pipette solution for biocytin staining Slice solution Triton X-100 Vectastain Elite ABC kit (Vector Labs) Equipment
Patch pipettes and other electrophysiology equipment Recording chamber (custom made) METHOD
1. Place the brain slices in a recording chamber (1.5 mL) and perfuse with slice solution. 2. Using two patch pipettes, load freshly prepared biocytin solution (pipette solution containing 5% biocytin hydrochloride) into each cell under study. For example, patch an interneuron with Adapted from Imaging in Neuroscience (ed. Helmchen and Konnerth). CSHL Press, Cold Spring Harbor, NY, USA, 2011. © 2014 Cold Spring Harbor Laboratory Press Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot083527
948
Downloaded from http://cshprotocols.cshlp.org/ at TULANE UNIV on September 8, 2014 - Published by Cold Spring Harbor Laboratory Press
Biocytin Staining
a biocytin-containing pipette, and then patch an astrocyte with another biocytin-containing pipette. 3. Without moving the patch pipettes, perfuse the slices with fixative for 10 min and then carefully withdraw the pipettes. 4. Postfix the slices for 10–12 h. Wash four times in 0.1 M PBS. 5. Incubate the slices in 0.1 M PBS containing 0.3% iodine at room temperature for 15 min. Wash 4× in PBS. 6. Incubate the slices in 0.1 M PBS containing 0.2% Triton X-100 and 0.2% albumin for 45 min at room temperature. Wash 4× in PBS. 7. Stain the fixed slices with the Vectastain Elite ABC kit. Mount with Permount.
DISCUSSION
We have used biocytin staining to confirm the identification of neural cells revealed by differential interference contrast (DIC) microscopy. Figure 1A illustrates a characteristic field of the stratum radiatum, containing both astrocytes and neurons, under DIC optics. Interneurons are characterized by large and bright somata (diameter 16.1 ± 2.7 µm; mean ± SD) with large dendrites, whereas astrocytic somata are small and round (diameter 8.1 ± 1.7 µm; mean ± SD) without large processes. The cell identified by DIC as an astrocyte was shown by biocytin staining (Fig. 1B) to have an extensive array of branched short processes, which are phenotypical for astrocytes. Nearby astrocytes could also be stained through gap junctions (Robinson et al. 1993). In contrast, the presumed interneuron was shown to have few evenly sized and less branched processes, a morphology that is stereotypical for neurons. The size of the biocytin-stained astrocyte seems larger than that of the same cell viewed under DIC optics. This might result from the mixture of short processes with the cell body. In this
FIGURE 1. Identification of interneurons and astrocytes in hippocampal slices. An astrocyte (white arrow; characterized by a small, rounded cell body and poorly defined, irregular processes) and an interneuron (red arrow; characterized by a large soma with large dendrites that project in all directions) located in the stratum radiatum were patched by two pipettes. After visualization under DIC optics (A), biocytin (0.5%) was injected into both cells to reveal cellular morphology (B). Scale bar, 10 µm. Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot083527
949
Downloaded from http://cshprotocols.cshlp.org/ at TULANE UNIV on September 8, 2014 - Published by Cold Spring Harbor Laboratory Press
J. Kang
experiment, a total of eight pairs of interneurons and astrocytes were injected with biocytin, and in all cases, biocytin staining confirmed cell identification under DIC.
RECIPES Fixative for Biocytin Staining
Add 40 mL of saturated picric acid (Sigma-Aldrich) and 80 mL of 25% glutaraldehyde (Fisher Scientific) to 9.88 mL of 0.2 M phosphate-buffered saline (Sigma-Aldrich). Dilute this solution 1:1 with 8% paraformaldehyde solution. Adjust the pH to 7.4 with NaOH. The final concentrations are 4% paraformaldehyde, 0.2% picric acid, and 0.1% glutaraldehyde. Paraformaldehyde Solution (8%)
Add 40 g of paraformaldehyde (Sigma-Aldrich) to 400 mL of distilled water. Heat to 60˚C and add 2–3 drops of 1 N NaOH. Adjust the final volume to 500 mL with distilled water. Store this stock solution at 4˚C. Pipette Solution for Biocytin Staining
Reagent Potassium gluconate KCl MgCl2 HEPES EGTA CaCl2 ATP Guanosine triphosphate (GTP) Glucose
Final concentration 120 mM 10 mM 1 mM 10 mM 0.1 mM 0.01 mM 1 mM 0.2 mM 4 mM
Adjust the pH to 7.2 with KOH. Slice Solution
Reagent NaCl KCl NaH2PO4 MgCl2 CaCl2 Glucose NaH2CO3
Final concentration 126 mM 2.5 mM 1.25 mM 2 mM 2 mM 10 mM 26 mM
The pH should be 7.4 when the solution is gassed with 5% CO2 and 95% O2 at room temperature (23˚C–24˚C).
REFERENCES Robinson SR, Hampson EC, Munro MN, Vaney DI. 1993. Unidirectional coupling of gap junctions between neuroglia. Science 262: 1072–1074.
950
Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot083527
