Extraction, Identification, and Quantification of Histones from Small Quantities of Specific Brain Tissue

Hassiba Beldjoud1, Fany Messanvi1, Nael Nadif Kasri2, Benno Roozendaal3

1 Department of Neuroscience, Section Anatomy, University Medical Center Groningen, Groningen, 2 Department of Human Genetics, Radboud University Medical Center, Nijmegen, 3 Donders Institute for Brain, Cognition, and Behaviour, Nijmegen
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 4.38
DOI:  10.1002/cpns.11
Online Posting Date:  July, 2016
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Histone posttranslational modifications (PTMs), by their action on the chromatin state, play a central role in the regulation of gene expression. The discovery that some PTMs in the brain are dynamically regulated by experience and environmental factors makes them an important subject for the study of plasticity changes in learning and memory, addiction, and psychiatric disorders. Current histone isolation protocols, however, require large amounts of tissue, which limits their application for analyzing small tissue samples from a specific brain region. We describe here a step‐by‐step protocol for histone extraction and isolation from 1 mm3 of tissue from brain punches, which allows reproducible and reliable results for histone PTM identification and quantification without losing anatomical precision. © 2016 by John Wiley & Sons, Inc.

Keywords: acetylation; epigenetics; histone; hippocampus; insular cortex; methylation; posttranslational modification; phosphorylation; western blotting

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Table of Contents

  • Introduction
  • Basic Protocol 1: Brain Isolation
  • Basic Protocol 2: Slice Preparation for Microdissection
  • Basic Protocol 3: Tissue Dissection and Histone Extraction
  • Basic Protocol 4: Histone Detection by Immunoblotting (Western Blotting)
  • Support Protocol 1: Stripping PVDF Membrane
  • Support Protocol 2: Analysis of Immunoblots
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Brain Isolation

  • Isopentane, ≥99% purity (e.g., Sigma Aldrich, cat. no. M32631)
  • Dry ice
  • Rodent of interest
  • Anesthetic (e.g., pentobarbital)
  • 250‐ml beaker
  • Thermometer capable of accurate measurements below −50°C
  • Vials or aluminum foil, for brain storage
  • Rodent guillotine
  • Surgical instruments, for brain removal
  • Round aluminum dishes with tabs
  • Forceps

Basic Protocol 2: Slice Preparation for Microdissection

  • Frozen brain ( protocol 1)
  • Freezing mounting medium (e.g., Leica Biosystems, FSC 22 Frozen Section Media)
  • Dry ice
  • Cryostat or freezing microtome
  • Paint brushes
  • Standard glass slides
  • Razor blade
  • Forceps

Basic Protocol 3: Tissue Dissection and Histone Extraction

  • Hypotonic lysis buffer (see recipe)
  • Tissue to be dissected (see protocol 2)
  • 0.2 N HCl
  • Trichloroacetic acid (TCA) precipitation solution (see recipe)
  • Acidified acetone: 100% acetone containing 0.1% (v/v) HCl
  • 100% acetone
  • 50 mM Tris⋅Cl (pH 8.0)/3% (w/v) SDS
  • Detergent‐compatible colorimetric assay kit (e.g., Bio‐Rad DC Protein Assay kit)
  • 5× Laemmli sample buffer (see recipe)
  • 50 mM Tris⋅Cl, pH 8.0
  • 4°C microcentrifuge
  • 1.5‐ml microcentrifuge tubes
  • 10‐ or 15‐cm petri dish
  • Palkovits punch technique brain punch set (0.75 to 1.25 mm; Stoelting, cat. no. 57401)
  • Dry ice
  • Rodent brain atlas
  • Tissue grinding pestle that fits 1.5‐ml microcentrifuge tubes (e.g., Sigma Aldrich, cat. no. Z359947‐100EA)
  • Vortex
  • Spectrophotometer
  • Heating block
CAUTION: Several of the reagents are highly corrosive (HCl, TCA, acetone). It is recommended to employ appropriate safety procedures (e.g., wearing gloves and protective clothing and working under a fume hood) and to follow the safety instructions on the manufacturers' data sheets.NOTE: All steps should be performed on ice, and all solutions and centrifuges should be chilled to 4°C prior to use.

Basic Protocol 4: Histone Detection by Immunoblotting (Western Blotting)

  • Histone samples (see protocol 3)
  • Discontinuous polyacrylamide gel
  • 100% methanol
  • Transfer buffer (see recipe)
  • LI‐COR blocking buffer
  • PBS (see recipe)
  • TBS (see recipe)
  • 5% (w/v) non‐fat milk
  • Appropriate primary and secondary antibodies
  • Vortex
  • Microcentrifuge
  • PVDF membrane
  • Forceps
  • Laboratory shaker
  • Odyssey IR Imaging System (LI‐COR Biosciences)
  • Additional reagents and equipment for gel electrophoresis (Gallagher, ) and transfer of immunoblots (unit 5.19)
NOTE: All equipment must be clean and rinsed with water prior to use. Only critical steps are mentioned in this protocol. For a more detailed description of the immunoblotting protocol see unit 5.19 (Gallagher et al., ).

Support Protocol 1: Stripping PVDF Membrane

  Additional Materials (also see protocol 4)
  • Stripping buffer (see recipe)
  • PBS (see recipe) with and without 0.1% (v/v) Tween 20

Support Protocol 2: Analysis of Immunoblots

  • Computer with LI‐COR Image Studio Software (available at https://www.licor.com/bio/products/software/)
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