A method for sectioning microwave-fixed brain prior to microdissection and acetylcholine analysis
D B Hoover1, E A Muth, D M Jacobowitz
1Laboratory of Clinical Science, National Institute of Mental Health, Building 10/Room 2D-46, Bethesda, MD 20014, USA.
Abstract:
Before proceeding with a detailed study of acetylcholine (ACh) distribution in the rat brain, a method was needed to make 300 microm brain sections for microdissection. We found cryostat sections of microwave-irradiated brains to be of poor quality, with rough surfaces and cracks. The use of a vibratome resulted in smooth, intact sections. This technique requires that unfrozen tissue be immersed in liquid during sectioning. Krebs-Ringer buffer or an inert fluorocarbon liquid (FC-47) were used for such sectioning. ACh was measured in forebrain regions from cryostat sections and vibratome sections cut in buffer or FC-47. Diffusion of ACh was apparent after sectioning in buffer, but not in FC-47. Vibratome sectioning in FC-47 should facilitate study of the distribution of ACh and other labile substances in rat brain.
Insights
A new vibratome sectioning method using FC-47 liquid preserves acetylcholine (ACh) distribution in rat brain sections, unlike cryostat or buffer methods.
Area of Science:
- Neuroscience
- Biochemistry
Background:
- Accurate measurement of neurotransmitter distribution in the brain requires high-quality tissue sections.
- Previous methods like cryostat sectioning of microwave-irradiated rat brains yielded poor quality sections unsuitable for microdissection.
Purpose of the Study:
- To develop and evaluate a reliable method for preparing 300 micrometer rat brain sections for microdissection and subsequent analysis of labile substances like acetylcholine (ACh).
Main Methods:
- Comparison of cryostat sectioning versus vibratome sectioning of unfrozen rat brains.
- Evaluation of different immersion liquids for vibratome sectioning: Krebs-Ringer buffer and an inert fluorocarbon liquid (FC-47).
- Measurement of acetylcholine (ACh) diffusion from brain sections prepared using different methods.
Main Results:
- Cryostat sections exhibited rough surfaces and cracks, compromising tissue integrity.
- Vibratome sectioning in Krebs-Ringer buffer led to apparent diffusion of ACh from the tissue.
- Vibratome sectioning in FC-47 resulted in smooth, intact sections with no detectable diffusion of ACh.
Conclusions:
- Vibratome sectioning of unfrozen rat brains immersed in FC-47 is a superior method for preserving the distribution of labile substances like ACh.
- This technique facilitates accurate microdissection and subsequent analysis of neurotransmitter distribution in the rat brain.
