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Using the Parafilm-assisted Microdissection (PAM) Method to Sample Rodent Nucleus Accumbens
Marco Pagliusi1, Arthur F Brandão1, Gabriel G Zanetti1
1Department of Structural and Functional Biology, University of Campinas, Campinas, Brazil.
Abstract:
Microdissection techniques are very important for anatomical and functional studies focused on neuroscience, where it is often necessary microdissect specific brain areas to perform molecular or anatomical analyses. The parafilm®-assisted microdissection (PAM) was previously described and involves the microdissection of tissue sections mounted on parafilm-covered glass slides. In this work, we describe the use of the PAM method to microdissect rodent nucleus accumbens (NAc). (1) We first describe the best way to perform the mouse euthanasia and how to remove the brain. (2) Next, we describe how to prepare the slides with parafilm® that will be used to receive the brain slices. (3) Following, we describe how to handle the brain in the cryostat, how to align the hemispheres and how to identify the NAc antero-posterior limits. (4) We also describe how to perform the staining and dehydration of the slices, a critical step to facilitate the microdissection and preserve macromolecules. (5) In the final step, we describe how to identify the dorso-ventral and latero-medial limits of the NAc and, finally, how to perform the manual microdissection of the area. This is a low-cost technique that allows the researcher to specifically microdissect any brain region, from which intact RNA and proteins can be extracted to perform several molecular analyses (e.g., real-time PCR, Western blot, and RNA-seq).
Insights
This study details a low-cost parafilm-assisted microdissection (PAM) method for isolating specific rodent brain regions, such as the nucleus accumbens (NAc). This technique enables precise tissue dissection for subsequent molecular and anatomical analyses.
Area of Science:
- Neuroscience
- Molecular Biology
- Anatomy
Background:
- Microdissection is crucial for neuroscience research, enabling molecular and anatomical analysis of specific brain regions.
- The parafilm-assisted microdissection (PAM) method offers a way to dissect tissue sections mounted on parafilm-covered glass slides.
Purpose of the Study:
- To describe the application of the PAM method for the microdissection of the rodent nucleus accumbens (NAc).
- To provide a detailed protocol for researchers to replicate the technique for isolating specific brain areas.
Main Methods:
- The protocol covers optimal mouse euthanasia and brain extraction.
- It includes slide preparation with parafilm®, brain handling in a cryostat, and identification of NAc boundaries.
- Staining, dehydration, and manual microdissection of the NAc are described in detail.
Main Results:
- The PAM method allows for the specific microdissection of the rodent nucleus accumbens.
- The described steps ensure proper tissue preparation and identification of anatomical landmarks.
- The technique facilitates the extraction of intact RNA and proteins from dissected tissues.
Conclusions:
- The parafilm-assisted microdissection (PAM) is a cost-effective technique for isolating specific brain regions in rodents.
- This method supports downstream molecular analyses, including real-time PCR, Western blot, and RNA-seq.
- PAM provides researchers with a reliable tool for precise anatomical dissection in neuroscience studies.
