Intracerebral Injections and Ultrastructural Analysis of High‐Pressure Frozen Brain Tissue

Marie‐Theres Weil1, Torben Ruhwedel2, Wiebke Möbius1, Mikael Simons3

1 Center Nanoscale Microscopy and Molecular Physiology of the Brain (CNMPB), Göttingen, 2 Department of Neurogenetics, Max‐Planck Institute for Experimental Medicine, Göttingen, 3 Munich Cluster for Systems Neurology (SyNergy), Munich
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 2.27
DOI:  10.1002/cpns.22
Online Posting Date:  January, 2017
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Abstract

Intracerebral injections are an invasive method to bypass the blood brain barrier and are widely used to study molecular and cellular mechanisms of the central nervous system. The administered substances are injected directly at the site of interest, executing their effect locally. By combining injections in the rat brain with state‐of‐the‐art electron microscopy, subtle changes in ultrastructure of the nervous tissue can be detected prior to overt damage or disease. The protocol presented here involves stereotactic injection into the corpus callosum of Lewis rats and the cryopreparation of freshly dissected tissue for electron microscopy. The localization of the injection site in tissue sections during the sample preparation for transmission electron microscopy is explained and possible artifacts of the method are indicated. With the help of this powerful combination of injections and electron microscopy, subtle effects of the applied substances on the biology of neural cells can be identified and monitored over time. © 2017 by John Wiley & Sons, Inc.

     
 
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Table of Contents

  • Significance Statement
  • Introduction
  • Strategic Planning (Optional)
  • Basic Protocol 1: Stereotactic Injection Into the Corpus Callosum of Lewis Rats
  • Basic Protocol 2: Cryopreparation of Lesion Sites for Electron Microscopy with High‐Pressure Freezing
  • Basic Protocol 3: Freeze Substitution of Brain Samples for Fixation and Embedding for Transmission Electron Microscopy
  • Basic Protocol 4: Ultramicrotomy of the Injection Site
  • Basic Protocol 5: Heavy Metal Staining of Thin Sections for Transmission Electron Microscopy
  • Basic Protocol 6: Locating Injection Site by Electron Microscopy
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Stereotactic Injection Into the Corpus Callosum of Lewis Rats

  Materials
  • Adult inbred female, 2‐ to 3‐month‐old Lewis rats (180 to 245 g; strain: LEW/crlcrlj, rat genome database, RGD ID: 631576, provided by Charles River) or equivalent
  • Isoflurane (Abbott, cat. no. 26675‐46‐2), store at room temperature
  • Anesthesia (see recipe)
  • Corn oil (Sigma, cat. no. C8267), store at room temperature
  • Sterile PBS (Gibco, cat. no. 10010‐015), store at room temperature
  • Monastral blue (Sigma, cat. no. 274011), 3% in water, filter with 0.45‐µm filter, store at room temperature
  • Sterile lubricant eye ointment (Delmed, cat. no. 01578681)
  • 70% ethanol (Sigma, cat. no. 32205)
  • Silkam (Braun, cat. no. 1048031)
  • Buprenorphine (0.03 mg/kg) (Temgesic, Animalcare), store at room temperature
  • Self‐built translucent plastic box
  • Injection system (World Precision Instruments, cat. no. 504127)
  • Microsyringe pump controller (World Precision Instruments model no. UMC4)
  • Stereotaxic frame
  • Scalpels (Swann Mortan, cat. no. 0301)
  • Permanent marker
  • Drill 105 (Dremel, cat. no. 26150105A)
  • Microdissection knife (Fine Science Tools, cat. no. 10056‐12)
  • Heat pad (cat. no. 12055/0200)

Basic Protocol 2: Cryopreparation of Lesion Sites for Electron Microscopy with High‐Pressure Freezing

  Materials
  • Liquid nitrogen
  • Injected rats (see protocol 1)
  • 5% isoflurane (Abbott, cat. no. 26675‐46‐2), store at room temperature
  • Superglue (Conrad, cat. no. TC‐SKP5G)
  • PBS (Gibco, cat. no. 10010‐015), ice cold
  • 4% PFA
  • 20% polyvinyl‐pyrrolidone (mol. wt. = 10,000; PVP in PBS) (Sigma, cat. no. P2307)
  • Hexadecane (Sigma, cat. no. 52270)
  • Leica HPM100 freezer (Leica, cat. no. 16HPM100EVN)
  • Scissors (Fine Science Tools, cat. no. 14024‐14)
  • Scalpels (Swann Mortan, cat. no. 0301)
  • Angled forceps or spoon
  • Tissue slicer (Zivic Labs, cat. no. BSMAA001‐1)
  • Platinum‐coated double‐edge razor blades (Science Services, cat. no. 72003‐01)
  • Leica vibratome VT1200S (Leica, cat. no. 1491200S001)
  • Fine brush (Electron Microscopy Sciences, cat. no. 66100‐03)
  • Pasteur pipets
  • Petri dishes
  • Harris cutting mat (Darmstadt, cat. no. 14222‐832)
  • Hollow biopsy punch (2‐mm diameter; World Precision Instruments, cat. no. 501817)
  • Fine‐tipped forceps
  • Specimen carriers, 3.0 × 0.5–mm diameter type A (Leica, cat. no. 16770141)
  • Specimen holder (Leica, cat. no. 16770135)
  • Filter paper (GE Healthcare, cat. no. 1001090)
  • Styrofoam box
  • Rubber‐coated forceps (Vomm, cat. no. 22SA ESD)
  • Cryovials (Sarstedt, cat. no. 72.380.992)

Basic Protocol 3: Freeze Substitution of Brain Samples for Fixation and Embedding for Transmission Electron Microscopy

  Materials
  • Tannic acid (Sigma, cat. no. 403040)
  • Glass‐distilled acetone (Electron Microscopy Sciences, cat. no. 10015)
  • Liquid nitrogen
  • High‐pressure frozen samples (see protocol 2)
  • Osmium tetroxide (OsO 4; Electron Microscopy Sciences, cat. no. 19130)
  • 5% uranyl acetate stock solution in methanol (SPI Chem, cat. no. 2624)
  • Epoxy resin (Epon; see recipe)
  • Leica aluminum tins equipped with disposable insets (flow‐through rings) (Leica, cat. no. 16707157)
  • Styrofoam box filled with liquid nitrogen
  • Rubber‐coated forceps (Vomm, cat. no. 22SA ESD)
  • Leica AFS2 (Leica, cat. no. 16707101)
  • Conical closures (Kapsto, Pöppelmann, cat. no. GPN 600 B300)
  • Plastic Pasteur pipets (VWR, cat. no. 612‐1684)
  • 50‐ml tubes (Falcon)
  • Needles
  • Parafilm‐covered glass slides
  • 60ºC incubator

Basic Protocol 4: Ultramicrotomy of the Injection Site

  Materials
  • Epon sample blocks (see protocol 3)
  • Chloroform (Merck, cat. no. 1.02445.1000)
  • Richardson's methylene blue/azure II blue (see recipe)
  • Eukitt mounting medium (O. Kindler, cat. no. 01015)
  • Leica Ultratrim (Leica, cat. no. 700138) or equivalent
  • Leica Ultracut S ultramicrotome (Leica, cat. no. 702501)
  • Diatome Histo diamond knife 6‐mm cutting length (Diatome, cat. no. DH4560)
  • Glass slides (Paul Marienfeld GmbH, cat. no. 0810000)
  • 60ºC hot plate
  • Phase contrast microscope
  • Diatome Ultra diamond knife 35º, 3.5‐mm cutting length (Diatome, cat. no. DU3535)
  • Formvar‐coated, 100‐mesh hexagonal copper grids (grids, Science Services, cat. no. 62010‐GU; formvar, Science Services, cat. no. 15800; see Slot and Geuze, ; Peters and Pierson, , for preparation)
  • Wooden stick with attached eyelash
  • Inverse forceps (World Precision Instruments, cat. no. 501205)
  • Grid storage box (Plano, cat. no. B801003050‐X)

Basic Protocol 5: Heavy Metal Staining of Thin Sections for Transmission Electron Microscopy

  Materials
  • 4% uranyl acetate in water (SPI Supplies, cat. no. 2624), filtered with a 0.22‐µm filter directly before use
  • 50‐nm sections collected on copper grids (see protocol 4)
  • 96‐well plate (Sigma, cat. no. M0812)
  • Parafilm
  • 1.5‐ml microcentrifuge tubes (Eppendorf)
  • Forceps
  • Filter paper (GE Healthcare, cat. no. 1001090)
  • Grid box (Plano, cat. no. B801003050‐X)
  • Lamp

Basic Protocol 6: Locating Injection Site by Electron Microscopy

  Materials
  • Electron microscope
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Figures

Videos

Literature Cited

Literature Cited
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