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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

This procedure describes the collection of discrete frozen brain regions to obtain high-quality protein and RNA using inexpensive and commonly available tools.

Abstract

As our understanding of neurobiology has progressed, molecular analyses are often performed on small brain areas such as the medial prefrontal cortex (mPFC) or nucleus accumbens. The challenge in this work is to dissect the correct area while preserving the microenvironment to be examined. In this paper, we describe a simple, low-cost method using resources readily available in most labs. This method preserves nucleic acid and proteins by keeping the tissue frozen throughout the process. Brains are cut into 0.5–1.0 mm sections using a brain matrix and arranged on a frozen glass plate. Landmarks within each section are compared to a reference, such as the Allen Mouse Brain Atlas, and regions are dissected using a cold scalpel or biopsy punch. Tissue is then stored at -80 °C until use. Through this process rat and mouse mPFC, nucleus accumbens, dorsal and ventral hippocampus and other regions have been successfully analyzed using qRT-PCR and Western assays. This method is limited to brain regions that can be identified by clear landmarks.

Introduction

This work illustrates the dissection of frozen brain regions for extraction of high-quality nucleic acid or protein using a reference, such as the Allen Mouse Brain Atlas1, as a guide. In this technique, brains are flash-frozen and stored at -80 °C for later sectioning and dissection while being maintained in a frozen condition. This process allows the researcher to harvest a large number of brains in one session and later dissect them for an accurate collection of multiple brain regions.

The accurate collection of brain regions of interest (ROIs) is often required when answering questions related to gene and pr....

Protocol

Animals used in this study were treated in an ethical and humane manner as set forth by Indiana University’s Institutional Animal Care and Use Committee (IACUC) and National Institutes of Health (NIH) guidelines.

NOTE: All tools and surfaces should be washed with an appropriate solvent to remove nucleases18 before starting any work.

1. Storing brains

  1. Quickly remove the brains from euthanized adult CD1 wildtype mice weighing .......

Representative Results

In order to validate this method, the medial prefrontal cortex was collected from adult CD1 wildtype male mice and RNA and protein were extracted and characterized. RNA was analyzed by capillary electrophoresis. Degraded RNA displays a loss in the intensity of the 28S and 18S ribosomal bands and also shows degradation products as a smear between 25 and 200 nucleotides (Figure 5A, sample 1). High quality RNA shows distinct ribosomal bands with little to no signal in the lower molecular weight.......

Discussion

This work describes a technique to isolate small, specific regions of brain while limiting degradation of nucleic acid and protein. Damage to brain tissues happens quickly once an organism dies. This is partially due to a rapid buildup of extracellular glutamate and the resultant excitotoxicity that occurs21. Messenger RNA is particularly vulnerable to degradation22,23. Breakdown of protein and nucleic acid is greatly reduced at low temper.......

Acknowledgements

This work was supported by the NIH, DA043982 and DA046196.

....

Materials

NameCompanyCatalog NumberComments
0.5 mm Mouse coronal brain matriceBraintree ScientificBS-SS 505CCutting block
0.5 mm Rat coronal brain matriceBraintree ScientificBS-SS 705CCutting block
1.0 mm Biopsy Punch with plungerElectron Microscopy Sciences69031-01
1.5 mL microcentrifuge tubesDot Scientific229443For storing frozen ROIs
1.5 mm Biopsy Punch with plungerElectron Microscopy Sciences69031-02
2.0 mm Biopsy Punch with plungerElectron Microscopy Sciences69031-03
4-12% NuPage gelInvitrogenNPO323BOXprotein gradient gel
Bioanalyzer SystemAgilent2100RNA analysis system
Dounce tissue grinderMillipore Sigma
D8938
Glass tissue homogenizer
Dry Ice
Fiber-LiteDolan-Jenner Industries Inc.Model 180Cool lamp
Glass platesLabRepCo11074010
HALTThermoFisher78440protease inhibitor cocktail
Low profile bladesSakura Finetek USA Inc.4689
mouse anti-actin antibodyDevelopmental Studies Hybridoma BankJLA20Antibody
NanodropThermo Scientific2000CUsed in initial RNA purity analysis
No. 15 surgical bladeSurgical Design Inc17467673
Odyssey Blocking bufferLiCor Biosciences927-40000Western blocking reagent
Omni Tissue Master 125VWR10046-866Tissue homogenizer
rabbit anti-KCC2 antibodyCell Signaling Technology94725SAntibody
RNA Plus Micro KitQiagen73034Used to extract RNA from small tissue samples
RNaseZapLife TechnologiesAM9780
Scalpel handleExcelta Corp.16050103
Standard razor bladesAmerican Line66-0362
TRIzol ReagentThermoFisher Scientific15596026Used to extract RNA from tissue

References

  1. . Allen Mouse Brain Atlas Available from: https://mouse.brain (2008)
  2. Kuleshova, E. P. Optogenetics - New Potentials for Electrophysiology. Neuroscience and Behavioral Physiology. 49 (2), 169-177 (2019).
  3. Scanziani, M., Häusser, M.

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