Home › Neuroscience › Laser Capture Microdissection - A Demonstration of the Isolation of Individual Dopamine Neurons and the Entire Ventral Tegmental Area
Neuroscience JoVE (Open Access) Citable · DOI

Laser Capture Microdissection - A Demonstration of the Isolation of Individual Dopamine Neurons and the Entire Ventral Tegmental Area

DOI: 10.3791/52336-v
What you'll learn
  • ✓ Prepare brain tissue and slides for laser capture microdissection
  • ✓ Operate infrared and ultraviolet laser systems for cell isolation
  • ✓ Isolate individual dopamine neurons from ventral tegmental area
  • ✓ Apply immunohistochemistry to identify and capture target neurons
Protocol

The isolation of individual dopamine neurons or the ventral tegmental area with direct or indirect immunohistochemistry is demonstrated using laser capture microdissection. Parameters for isolation of tissue from a glass slide using an infrared laser and from membrane slides using the combination of an infrared and ultraviolet laser are discussed.

Difficulty
advanced
Total time
~4–6 hours per animal (including tissue preparation, sectioning, immunostaining, and microdissection)
Model organism
Mouse (brain tissue)
Biosafety
BSL-1

Steps

1
Prepare slides and tissue for laser capture microscopy

Mount cryostat sections on appropriate slides (glass or PEN membrane) and apply direct or indirect immunohistochemistry to label dopamine neurons. Ensure tissue adhesion and staining quality before microdissection.

▶ 01:12
2
Set up laser capture microdissection system

Configure the infrared and ultraviolet laser parameters for isolation. Mount the prepared slide on the microscope stage and align optical and laser systems for accurate cell targeting.

▶ 03:08
3
Capture ventral tegmental area from PEN membrane slides

Use combined infrared and ultraviolet lasers to isolate the entire ventral tegmental area region from the membrane slide. Execute precise laser cuts to separate tissue from the slide.

▶ 05:19
4
Isolate individual dopamine neurons using laser capture

Target and capture immunostained individual dopamine neurons from glass slides using infrared laser. Collect isolated neurons in collection tubes for downstream molecular analysis.

▶ 06:19
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