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UNIT 2.27 Intracerebral Injections and Ultrastructural Analysis of High-Pressure Frozen Brain Tissue

  1. Marie-Theres Weil1,2,3,
  2. Torben Ruhwedel2,
  3. Wiebke Möbius2,3,
  4. Mikael Simons1,4,5,6

Published Online: 3 JAN 2017

DOI: 10.1002/cpns.22

Current Protocols in Neuroscience

Current Protocols in Neuroscience

How to Cite

Weil, M.-T., Ruhwedel, T., Möbius, W., and Simons, M. 2017. Intracerebral injections and ultrastructural analysis of high-pressure frozen brain tissue. Curr. Protoc. Neurosci. 78:2.27.1-2.27.18. doi: 10.1002/cpns.22

Author Information

  1. 1

    Department of Cellular Neuroscience, Max-Planck Institute for Experimental Medicine, Göttingen, Germany

  2. 2

    Department of Neurogenetics, Max-Planck Institute for Experimental Medicine, Göttingen, Germany

  3. 3

    Center Nanoscale Microscopy and Molecular Physiology of the Brain (CNMPB), Göttingen, Germany

  4. 4

    Institute of Neuronal Cell Biology, The Technical University of Munich, Munich, Germany

  5. 5

    German Center for Neurodegenerative Disease (DZNE), Munich, Germany

  6. 6

    Munich Cluster for Systems Neurology (SyNergy), Munich, Germany

Publication History

  1. Published Online: 3 JAN 2017

Significance Statement

  1. Top of page
  2. Significance Statement
  3. Introduction
  4. Strategic Planning (Optional)
  5. Basic Protocol 1: Stereotactic Injection Into the Corpus Callosum of Lewis Rats
  6. Basic Protocol 2: Cryopreparation of Lesion Sites for Electron Microscopy with High-Pressure Freezing
  7. Basic Protocol 3: Freeze Substitution of Brain Samples for Fixation and Embedding for Transmission Electron Microscopy
  8. Basic Protocol 4: Ultramicrotomy of the Injection Site
  9. Basic Protocol 5: Heavy Metal Staining of Thin Sections for Transmission Electron Microscopy
  10. Basic Protocol 6: Locating Injection Site by Electron Microscopy
  11. Reagents and Solutions
  12. Commentary
  13. Literature Cited

Intracerebral injections are used to deliver molecules into the brain to study the molecular and cellular mechanisms within the central nervous system at the site of interest. Electron microscopy is a useful tool to visualize the effects at the ultrastructural level. In this protocol, intracerebral injections into rat brains are combined with a preparation technique for electron microscopy that results in superior ultrastructural preservation. Cryopreparation by high-pressure freezing and freeze-substitution for electron microscopy is the state-of-the-art method to study ultrastructure with best possible preservation. Together, this powerful tool allows one to assess the effect of administered substances on a variety of neural cells with close-to-native ultrastructural preservation.

Introduction

  1. Top of page
  2. Significance Statement
  3. Introduction
  4. Strategic Planning (Optional)
  5. Basic Protocol 1: Stereotactic Injection Into the Corpus Callosum of Lewis Rats
  6. Basic Protocol 2: Cryopreparation of Lesion Sites for Electron Microscopy with High-Pressure Freezing
  7. Basic Protocol 3: Freeze Substitution of Brain Samples for Fixation and Embedding for Transmission Electron Microscopy
  8. Basic Protocol 4: Ultramicrotomy of the Injection Site
  9. Basic Protocol 5: Heavy Metal Staining of Thin Sections for Transmission Electron Microscopy
  10. Basic Protocol 6: Locating Injection Site by Electron Microscopy
  11. Reagents and Solutions
  12. Commentary
  13. Literature Cited

To study the ultrastructure of cells within a tissue, one crucial tool is electron microscopy. Improvements in fixation methods are now allowing scientists to study the ultrastructure of cellular structures within the brain with unprecedented detail and reliability. Here, a protocol to perform cryopreparation after intracerebral injections of substances into the rat brain is presented. Compared to conventionally prepared samples obtained by aldehyde-fixation and subsequent dehydration, cryopreparation preserves the ultrastructure of the sample as close as possible to the native state (Korogod et al., 2015). By combining this state-of-the-art method of sample preparation with intracerebral injections, a direct examination of the local effect of the administered injected solution (chemicals, drugs for treatment, antibodies) on the ultrastructure of all various cell types within the affected region of the CNS is possible. This method was successfully applied to investigate the mechanisms of myelin breakdown in demyelinating diseases by using, e.g., injections of antibodies and complement (Weil et al., 2016). The fixation methods described in this protocol turned out to be crucial for the observation of subtle and early changes in myelin architecture during demyelination.

Strategic Planning (Optional)

  1. Top of page
  2. Significance Statement
  3. Introduction
  4. Strategic Planning (Optional)
  5. Basic Protocol 1: Stereotactic Injection Into the Corpus Callosum of Lewis Rats
  6. Basic Protocol 2: Cryopreparation of Lesion Sites for Electron Microscopy with High-Pressure Freezing
  7. Basic Protocol 3: Freeze Substitution of Brain Samples for Fixation and Embedding for Transmission Electron Microscopy
  8. Basic Protocol 4: Ultramicrotomy of the Injection Site
  9. Basic Protocol 5: Heavy Metal Staining of Thin Sections for Transmission Electron Microscopy
  10. Basic Protocol 6: Locating Injection Site by Electron Microscopy
  11. Reagents and Solutions
  12. Commentary
  13. Literature Cited

For stereotactic injections, an adequate control should be used, such as injection with only the vehicle. All injections should be done on the same day as all of the animals must be processed by high-pressure freezing on the same day. For freeze substitution, it must be taken into account that only 30 samples can be processed at one time and that the protocol requires 6 days. Samples of one experiment (control and treatment) should be processed in one round of freeze substitution in order to keep them as comparable as possible. A flowchart of all preparation steps is provided in Figure 2.27.1.

figure

Figure 2.27.1. Flowchart of intracerebral injections and ultrastructural analysis of high-pressure frozen brain tissue. Substances are administered into a living animal by stereotactic injections into the corpus callosum. At the time point of interest, the lesion site is cryopreserved by high-pressure freezing and subsequent freeze substitution. Semi-thin sections are cut and stained to identify the lesion site within the samples. Following trimming, samples are processed for electron microscopy by ultrathin sectioning and heavy metal staining. After locating the injection site in the transmission electron microscope, micrographs can be acquired and analyzed.

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

  1. Top of page
  2. Significance Statement
  3. Introduction
  4. Strategic Planning (Optional)
  5. Basic Protocol 1: Stereotactic Injection Into the Corpus Callosum of Lewis Rats
  6. Basic Protocol 2: Cryopreparation of Lesion Sites for Electron Microscopy with High-Pressure Freezing
  7. Basic Protocol 3: Freeze Substitution of Brain Samples for Fixation and Embedding for Transmission Electron Microscopy
  8. Basic Protocol 4: Ultramicrotomy of the Injection Site
  9. Basic Protocol 5: Heavy Metal Staining of Thin Sections for Transmission Electron Microscopy
  10. Basic Protocol 6: Locating Injection Site by Electron Microscopy
  11. Reagents and Solutions
  12. Commentary
  13. Literature Cited

The purpose of this method is to examine the effect of the administered agent (e.g., demyelinating chemicals, viruses, antibodies, drugs, siRNA, etc.) at the ultrastructural level in the affected brain region (see Fig. 2.27.2). This protocol describes the delivery of substances into the corpus callosum of anesthetized rats, but by changing the coordinates it can be applied for other brain regions as well. Upon complete anesthesia (animals were unresponsive to pinching of the paws), the rats are mounted onto a stereotaxic frame. After determining the correct location of the injection site (using the rat brain atlas described in Paxinos and Watson, 2007) according to the position of the bony features bregma and lambda on the skull as reference points, a hole is drilled into the skull to gain access to the brain. The injection of the drug of interest is performed very slowly and carefully so as not to disrupt the tissue structure. After injection, the animal is sutured and allowed to recover. Post-operative care should be taken by treating the rats with analgesia and examining them for pain.

figure

Figure 2.27.2. Location of bony features of the rat skull. Stereotactic landmarks, such as the bregma or lambda on the skull of the rat are used to define the precise coordinates to achieve a reproducible injection site. The approximate injection site to target the corpus callosum is marked with a red “x”.

NOTE: All protocols using live animals must first be reviewed and approved by an Institutional Animal Care and Use Committee (IACUC) and must follow officially approved procedures for the care and use of laboratory animals.

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)
  • 1.

    Place rat in a translucent plastic box filled with 5% isoflurane (in oxygen). Wait until the animal stops moving, then remove the rat and inject it with ketamine/xylazine (see step 2).

  • 2.

    Anesthetize the rat by intraperitoneal (i.p.) injection of ketamine (60 mg/kg body weight) and xylazine (8 mg/kg body weight).

    Animal will remain anesthetized for 2 to 3 hr.

  • 3.

    While waiting for complete anesthesia of the animal, prepare the injection capillary by mounting it to the injection system following the manufacturer's instructions. Fill the capillary with corn oil to achieve a smooth release of the injection solution. Ensure that injection needle of microsyringe pump controller is inserted into the capillary at least halfway to ensure a stable injection system. Wash capillary two times with sterile PBS before loading the capillary with the injection solution. Then load the capillary with the injection solution containing a 1:25 dilution of 3% monastral blue to identify the lesion site for downstream processing.

  • 4.

    Apply sterile lubricant eye ointment on the eyes of the rat to prevent them from drying out.

  • 5.

    Test reflexes of the animal carefully before starting the procedure.

  • 6.

    Immobilize the animal using a stereotactic frame by fixing the head with the bars in the ears, putting just enough pressure to keep it stable. Place teeth of the rat on the mouth rest and adjust the height in such a way that the skull of the animal is straight. Make sure that the tongue of the animal is pulled out to prevent suffocation.

    To avoid hurting the animal, make sure not to over tighten the bars.

  • 7.

    Wipe head of rat with 70% ethanol. Using a scalpel, cut the skin of the animal open from just behind the eyes to the lateral incisure (see Fig. 2.27.2).

  • 8.

    First, determine coordinates of the bregma (see Fig. 2.27.2) by placing the capillary of the injection system above it. Bregma determines the zero-point in x- and z- directions. Then, move the capillary 1 mm caudally and 2 mm laterally relative to the bregma and lower it in close proximity to the skull to determine the position where the hole should be drilled. Mark the position of the capillary on the skull with a cross using a permanent marker. Raise the capillary up enough so there is space to drill the hole.

    This protocol can be used on mice by adapting the coordinates according to the mouse brain atlas (Franklin and Paxinos, 2008).

  • 9.

    Carefully drill a small hole through the skull at low speed by hand. Do not damage the brain by placing too much pressure or drilling too deep. Drill a hole big enough for the capillary (∼1 to 2 mm). To prevent damage to the brain, carefully remove the last thin layer of cartilage and meninges with a microdissection knife.

  • 10.

    Slowly lower the capillary to the brain until the capillary touches the brain surface, which is observed by a change in reflection (this indicates the zero-position in y direction). To target the corpus callosum, manually lower the needle slowly by 3 mm (∼1 mm per 15 sec).

  • 11.

    Inject 1 µl of the injection mix at a speed of 300 nl/min. Leave the needle in place for 5 min before slowly retracting it.

  • 12.

    Suture the skin with Silkam using single stitches and place the rat in a cage on a heat pad at ∼35ºC until fully awake.

  • 13.

    To ensure that the animal does not suffer from post-operative pain, inject analgesia buprenorphine (0.03 mg/kg) i.p. directly after surgery and repeat two times after 6 and 12 hr p.i. Keep animals on a heat pad to avoid hypothermia and check regularly (every 30 min) for signs of pain.

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

  1. Top of page
  2. Significance Statement
  3. Introduction
  4. Strategic Planning (Optional)
  5. Basic Protocol 1: Stereotactic Injection Into the Corpus Callosum of Lewis Rats
  6. Basic Protocol 2: Cryopreparation of Lesion Sites for Electron Microscopy with High-Pressure Freezing
  7. Basic Protocol 3: Freeze Substitution of Brain Samples for Fixation and Embedding for Transmission Electron Microscopy
  8. Basic Protocol 4: Ultramicrotomy of the Injection Site
  9. Basic Protocol 5: Heavy Metal Staining of Thin Sections for Transmission Electron Microscopy
  10. Basic Protocol 6: Locating Injection Site by Electron Microscopy
  11. Reagents and Solutions
  12. Commentary
  13. Literature Cited

After successful intracerebral injection, the animals are processed for electron microscopy after appropriate time points (see Fig. 2.27.1). To achieve the state-of-the-art preservation of the ultrastructure of the brain in a close-to-native state, this protocol employs high-pressure freezing of the injection site.

After culling the animal, the brain is quickly removed and trimmed around the injection site with a razor blade. Vibratome sections of the brain are cut and the injection site is punched out of the sections using a biopsy punch. After carefully loading into the specimen carrier, the sample is frozen with liquid nitrogen under high pressure (∼2000 bar) using a high-pressure freezer, e.g., a Leica HPM100. The samples are then stored in liquid nitrogen until future processing by freeze substitution.

Materials
  • Liquid nitrogen
  • Injected rats (see Basic 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)
  • 1.

    Cool down the Leica HPM100 machine by filling the internal storage dewar with liquid nitrogen to 100%. Wait 20 min and refill. Allow the whole system to cool down for an additional 30 min before carrying out a few test freezing cycles and freezing the first samples.

  • 2.

    Anesthetize a rat with 5% isoflurane (in oxygen). After complete anesthesia (as indicated by the absence of any reflex), cut the head off with sharp, strong scissors or guillotine.

  • 3.

    Using a scalpel, cut the skin from the nose to the neck and flip the skin to either side.

  • 4.

    Cut the skull from the brainstem to the coronal suture with a pair of strong scissors. Make a lateral incision to either side and remove the part of the skull covering the brainstem.

  • 5.

    Use strong scissors to cut the skull on either side of the brain to the ears and eyes. Cut the bone between the eyes. Remove the bone by flapping to either side.

  • 6.

    Gently remove the brain from the skull with a spoon or angled forceps by inserting the tool beneath the brain and gently pulling the brain out. Place the brain into a tissue slicer.

  • 7.

    Cut the brain coronally with a razor blade ∼2 mm away from the injection site (indicated by the blue dye) towards the olfactory bulb and ∼5 mm towards the brainstem. Cut away the uninjected hemisphere.

    The other hemisphere may also be used as a control by injecting vehicle only, but care must be taken so that the injected substances do not spread towards the other hemisphere.

  • 8.

    Use superglue to mount the flat-sided tissue where the brainstem was cut off onto the vibratome plate. Allow glue to harden for ∼30 sec. In the meantime, fill the buffer tray with ice-cold PBS and place it into the ice bath. Then, immerse the plate with the attached brain into the buffer tray. Attach a razor blade to the head of the vibratome and set the following parameters: 200-µm slice thickness; 1-mm amplitude; speed of 0.6 to 0.7 mm/sec.

  • 9.

    Orient the brain in such a way that the cortex faces the razor blade. Set the cutting window on the vibratome. Begin cutting with the indicated settings.

  • 10.

    Gently hold the top of the tissue with a fine brush and push a stream of cold buffer from a Pasteur pipet towards the razor blade to help cut equally thick sections. Completely cut the injection site. Fix all sections not needed for cryopreparation for histology using 4% PFA or for conventional electron microscopy.

  • 11.

    Collect and align the sections one by one in the cutting chamber, taking care not to touch the area of interest. Select two sections of good quality (assess quickly with a dissection microscope) that contain monastral blue as marker for the injection site.

    The sections do not necessarily have to be adjacent, although it is preferred, but should contain the region of interest.

  • 12.

    Transfer these two sections into a petri dish containing ice-cold PBS and then transfer the sections with a fine brush onto a cutting mat with ∼0.5 ml of 20% PVP in PBS. Use a 2-mm hollow biopsy punch and place it above the middle of the injection site. Punch out the tissue and gently rotate to cut and detach the tissue inside the punch. Remove the tissue piece by pushing it out of the biopsy punch. If the punched tissue is stuck to the plastic mat, gently remove the tissue with fine-tipped forceps by only touching the rim of the tissue. Submerge the specimen carrier in the droplet of 20% PVP and transfer the tissue into the carrier by placing the specimen carrier underneath the tissue and moving it up. Only touch the edge of the tissue if necessary. Remove air bubbles and transfer to specimen holder into the middle plate on top of the lower cylinder half. Close the specimen carrier with a second carrier wet on the flat side with hexadecane and then, remove excess liquid using filter paper. Close the specimen sandwich by lowering the upper cylinder half.

  • 13.

    Push the button to freeze the sample and remove the sample from the sample dewar. Rapidly transfer the specimens into a Styrofoam box filled with liquid nitrogen.

  • 14.

    Remove the specimen carrier from the middle plate and remove the lid of the carrier. Transfer the specimen carrier containing the sample with rubber-coated forceps to perforated cryovials inside the Styrofoam box filled with liquid nitrogen. Store samples in liquid nitrogen until the freeze substitution is performed.

    The cryovials are perforated on the upper half with a hot (heated) needle to prevent the vial from floating in the liquid nitrogen and they are labeled with the sample name.

    The samples can be stored in liquid nitrogen for several months (up to years), but avoid ice contamination. It is recommended to process samples within a shorter time frame (up to several weeks).

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

  1. Top of page
  2. Significance Statement
  3. Introduction
  4. Strategic Planning (Optional)
  5. Basic Protocol 1: Stereotactic Injection Into the Corpus Callosum of Lewis Rats
  6. Basic Protocol 2: Cryopreparation of Lesion Sites for Electron Microscopy with High-Pressure Freezing
  7. Basic Protocol 3: Freeze Substitution of Brain Samples for Fixation and Embedding for Transmission Electron Microscopy
  8. Basic Protocol 4: Ultramicrotomy of the Injection Site
  9. Basic Protocol 5: Heavy Metal Staining of Thin Sections for Transmission Electron Microscopy
  10. Basic Protocol 6: Locating Injection Site by Electron Microscopy
  11. Reagents and Solutions
  12. Commentary
  13. Literature Cited

Cryopreparation yields samples that are quickly and physically immobilized in a close-to-native and fully hydrated state, as it transforms water into amorphous ice. To image these samples at room temperature by electron microscopy, the water must be replaced with organic solvents while stabilizing and fixing the structures at low temperature. The semi-automated freeze substitution protocol described here requires loading the samples into insets of aluminum tins that are placed into a chamber of the Leica AFS 2. The dehydration and fixation is performed at −90ºC by using different cocktails containing tannic acid followed by osmium tetroxide and uranyl acetate to stabilize and contrast the specimen. After slowly warming up to 4ºC, the samples are embedded in epoxy resin by infiltrating with increasing amounts of epoxy in acetone (modified from Siksou et al., 2007). After embedding the samples using gelatin capsules, the resin is polymerized for 24 hr.

CAUTION: Osmium tetroxide, uranyl acetate, and some components of the epoxy resin are toxic or carcinogen and should be handled in a fume hood with great care and appropriate personal protection (nitrile gloves, safety goggles, and laboratory coat).

Materials
  • Tannic acid (Sigma, cat. no. 403040)
  • Glass-distilled acetone (Electron Microscopy Sciences, cat. no. 10015)
  • Liquid nitrogen
  • High-pressure frozen samples (see Basic Protocol 2)
  • Osmium tetroxide (OsO4; 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
  • 1.

    Prepare fresh 0.1% tannic acid in glass-distilled acetone and use this solution to fill Leica aluminum tins equipped with disposable insets suitable for ten specimens (flow-through rings) until the insets are half-full. To identify position 1 in the disposable inset, cut a v-shape at the upper rim of one slot prior to use. Place tins into liquid nitrogen to freeze the tannic acid/acetone-mix.

    The samples can be freeze substituted at any time after cryofixation. Ideally, the samples should be processed quickly after the high pressure freezing, but can also be stored in liquid nitrogen for a prolonged time.

  • 2.

    Transfer samples from the liquid nitrogen container into a Styrofoam box filled with liquid nitrogen. Remove the specimen carriers out of the cryovial in liquid nitrogen; then, using rubber-coated forceps, place them upside down into the aluminum tin insets filled with frozen tannic acid/acetone-mix.

    Do not thaw samples and make sure that the tips of the forceps are always in liquid nitrogen before touching samples and avoid liquid nitrogen spillage into the tannic acid/acetone-mix.

  • 3.

    Quickly move tins from liquid nitrogen into the Leica AFS 2 pre-cooled to −90ºC. Cover tins with disposable plastic lids. Then, freeze-substitute samples in a semi-automated fashion according to the protocol shown in Table 2.27.1.

Table 2.27.1. Automated Freeze Substitution Protocol (Modified from Siksou et al., 2007)
SolutionDurationTemperature
0.1% tannic acid in acetone24 hr−90°C
Ice-cold acetone4 × 30 min−90°C
2% osmium tetroxide, 0.1% uranyl acetate in acetone7 hr−90°C
2% osmium tetroxide, 0.1% uranyl acetate in acetone14 hrWarm up to −20°C, in 5°C/hr
2% osmium tetroxide, 0.1% uranyl acetate in acetone16 hr−20°C
2% osmium tetroxide, 0.1% uranyl acetate in acetone2.4 hrWarm up to 4°C, in 10°C/hr
2% osmium tetroxide, 0.1% uranyl acetate in acetone1 hr4°C
Cold acetone4 × 30 min4ºC
Cold acetone1 hrRoom temperature
Acetone/Epon mix (2:1)2 hrRoom temperature
Acetone/Epon mix (1:1)2 hrRoom temperature
Acetone/Epon mix (1:2)2 hrRoom temperature
90% Epon in acetoneOvernightRoom temperature
Fresh pure EponOvernightRoom temperature
  • 4.

    After 24 hr incubation with tannic acid at −90°C, remove tannic acid by aspirating liquid with a plastic Pasteur pipet and replacing it with acetone pre-cooled to −90ºC. Perform four washes, 30 min each wash. Ensure that the samples are always covered with liquid.

  • 5.

    During the fourth wash, prepare the contrasting mix of osmium tetroxide and 5% uranyl acetate in acetone. Open an ampule with 0.5 g osmium tetroxide (OsO4) crystals in the fume hood, wearing a laboratory coat, gloves, and mask, and place contents into a 50-ml tube. Add 25 ml glass-distilled acetone. Use plastic pasteur pipets to dissolve OsO4 crystals in acetone. After the OsO4 is completely dissolved, add 500 µl of 5% uranyl acetate stock solution in methanol to achieve a final concentration of 2% OsO4 and 0.1% uranyl acetate. Carefully transfer the mix into closable glass vials that fit into the Leica AFS2 chamber using a Pasteur pipet. Then, transfer the closed vials into the Leica AFS2 to cool down during the fourth wash. Then, incubate the samples in the contrasting cocktail following the programmed temperature curve (see Table 2.27.1; 7 hr at −90ºC, 14 hr temperature raise to −20ºC with an increment of 5ºC/hr, 16 hr at −20ºC followed by a 2.4-hr temperature raise of 10ºC/hr to 4ºC). Carefully dispose of the waste (including waste from the washing steps) according to appropriate regulations.

    The samples turn black after incubation with the contrasting cocktail.

  • 6.

    After reaching a temperature of 4ºC, remove tins from the AFS2 chamber and place on ice in a fume hood. Wash samples four times with ice-cold acetone (carefully removing and disposing liquid waste according to appropriate regulations). Allow samples to warm up to room temperature over 1 hr prior to infiltration.

  • 7.

    Infiltrate samples with increasing concentrations of Epon in acetone (2:1, 1:1, 1:2 acetone/Epon), incubating 2 to 3 hr for each step. Infiltrate samples with 90% Epon in acetone overnight at room temperature.

  • 8.

    Infiltrate samples two times with fresh Epon resin over 4 hr. Carefully remove the freeze-substituted tissue from the sample carrier with the help of a bent needle. Place samples onto Parafilm-covered glass slides with Epon-filled gelatin capsules turned upside down on the sample. Include labels in the resin to identify samples.

  • 9.

    Polymerize Epon resin 24 hr at 60ºC.

  • 10.

    Remove the gelatin capsule by boiling the resin blocks in water for 15 min.

Basic Protocol 4: Ultramicrotomy of the Injection Site

  1. Top of page
  2. Significance Statement
  3. Introduction
  4. Strategic Planning (Optional)
  5. Basic Protocol 1: Stereotactic Injection Into the Corpus Callosum of Lewis Rats
  6. Basic Protocol 2: Cryopreparation of Lesion Sites for Electron Microscopy with High-Pressure Freezing
  7. Basic Protocol 3: Freeze Substitution of Brain Samples for Fixation and Embedding for Transmission Electron Microscopy
  8. Basic Protocol 4: Ultramicrotomy of the Injection Site
  9. Basic Protocol 5: Heavy Metal Staining of Thin Sections for Transmission Electron Microscopy
  10. Basic Protocol 6: Locating Injection Site by Electron Microscopy
  11. Reagents and Solutions
  12. Commentary
  13. Literature Cited

To examine the samples at high resolution by electron microscopy, ultrathin sections of the polymerized plastic blocks must be prepared with a section thickness of ∼50 nm. As ultrathin sectioning is technically challenging but crucial to achieve good results, the following brief protocol is intended for experienced users.

As a first step, the blocks containing the samples must be trimmed around the tissue. Semi-thin sections of 500-nm thickness are cut until the complete sample is contained within the sections. To localize the region of interest, the semi-thin sections are stained with Richardson's methylene blue/azure II blue. Where after, the block is further trimmed around the injection site, depending on the scientific question. Subsequently, 50-nm ultrathin sections are cut and collected on copper grids that can be contrasted and visualized by transmission electron microscopy.

Materials
  • Epon sample blocks (see Basic 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, 2007; Peters and Pierson, 2008, for preparation)
  • Wooden stick with attached eyelash
  • Inverse forceps (World Precision Instruments, cat. no. 501205)
  • Grid storage box (Plano, cat. no. B801003050-X)
  • 1.

    Trim Epon blocks around the tissue sample using a Leica Ultratrim (or equivalent or razor blades).

  • 2.

    Cut block into semi-thin sections of 500 nm thickness using a Leica Ultracut S ultramicrotome and a Histo diamond knife (or equivalent). Straighten the sections with chloroform vapor.

    Semi-thin sections reflect rainbow colors after cutting.

  • 3.

    Collect the semi-thin sections onto glass slides and dry them on a hot plate at 60ºC.

  • 4.

    Stain samples on the glass slides with Richardson's methylene blue/azure II solution for ∼1 min at 60ºC (Richardson et al., 1960). Collect staining solution into a waste container and dispose according to local regulations. Rinse the glass slides for ∼30 sec under running tap water. Mount slides with Eukitt mounting medium and allow them to dry overnight at room temperature in a fume hood.

  • 5.

    Locate the structure of interest and the injection site (identified by the tracer dye, the needle track in early injections, or the accumulation of cells in later stage lesions) under a phase contrast microscope (see Fig. 2.27.3).

    The blue staining of the Richardson's methylene blue/azure II solution stains lipid-rich areas, such as the corpus callosum. The nuclei of cells are also stained so that the injection site is apparent due to an accumulation of (immune) cells.

figure

Figure 2.27.3. Localization of injection sites in semi-thin sections. Representative images of semi-thin sections (500-nm) of injections in the corpus callosum of adult Lewis rats stained with Richardson's methylene blue/azure II blue. (A) Image shows a semi-thin section prior to trimming for sectioning for transmission electron microscope, whereas (B) shows the same sample after trimming. (C) Image displays a sample where the injection site can be clearly localized as shown by the monastral blue marker. The red boxes on the images on left panels (A, C, E) are shown magnified in right panels (B, D, F). The accumulation of cells in (E) indicates the lesion site. Scale bar for (A, C, E): 0.5 mm, for (B, D, F): 150 µm. The corpus callosum is labeled with CC, whereas the border to the cortex is marked by a green dotted line.

  • 6.

    After localizing the region of interest on the semi-thin sections, further trim the block around the injection site to facilitate its detection during imaging by the electron microscope.

    This step must be carried out carefully to avoid unintended loss of tissue.

  • 7.

    Cut and stain semi-thin sections of 500-nm thickness of the trimmed block as described above in steps 2 to 6.

    It is helpful to obtain light microscopy images of the injection site and have them available when searching for the region of interest under the electron microscope.

  • 8.

    Cut ultrathin sections of 50-nm thickness of the trimmed block using a Leica Ultracut S ultramicrotome and an Ultra 35 diamond knife (or equivalent). Straighten the sections using chloroform vapor.

    Ultrathin sections of 50-nm thickness appear grey (<60 nm) due to their interference pattern. If the sections appear golden, then they are too thick (>90 nm).

  • 9.

    Collect the ultrathin sections on copper grids with the help of inverse forceps and an eyelash attached to a wooden stick. Leave the grids to dry before storing in the grid box.

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

  1. Top of page
  2. Significance Statement
  3. Introduction
  4. Strategic Planning (Optional)
  5. Basic Protocol 1: Stereotactic Injection Into the Corpus Callosum of Lewis Rats
  6. Basic Protocol 2: Cryopreparation of Lesion Sites for Electron Microscopy with High-Pressure Freezing
  7. Basic Protocol 3: Freeze Substitution of Brain Samples for Fixation and Embedding for Transmission Electron Microscopy
  8. Basic Protocol 4: Ultramicrotomy of the Injection Site
  9. Basic Protocol 5: Heavy Metal Staining of Thin Sections for Transmission Electron Microscopy
  10. Basic Protocol 6: Locating Injection Site by Electron Microscopy
  11. Reagents and Solutions
  12. Commentary
  13. Literature Cited

To visualize the ultrastructure by transmission electron microscopy, the contrast within the sample must be further enhanced in the ultrathin sections by so-called post-staining. Here, heavy metals (such as uranyl acetate or lead citrate) are deposited onto macromolecules to induce scattering of the electrons and generate the contrast.

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 Basic 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
  • 1.

    Cover a 96-well plate with Parafilm and press over the Parafilm with the end of a 1.5-ml microcentrifuge tube (this leads to small pits in each well). Place a small drop of 4% filtered uranyl acetate onto the Parafilm.

  • 2.

    Using forceps, place three copper grids per sample upside down onto the contrasting solution. Dependent on the type of tissue, incubate the sample 15 to 30 min in the dark.

  • 3.

    Place four drops of water into the adjacent wells and transfer the grids after contrasting one-by-one from one drop of water to the next.

  • 4.

    Remove the grids from the last droplet of water and dry them by holding a piece of filter paper at the edge of the grid.

  • 5.

    Place the grid into a grid box and place the open box under a lamp to completely dry.

  • 6.

    After 5 to 10 min, close the box and store at room temperature until imaging under an electron microscope.

Basic Protocol 6: Locating Injection Site by Electron Microscopy

  1. Top of page
  2. Significance Statement
  3. Introduction
  4. Strategic Planning (Optional)
  5. Basic Protocol 1: Stereotactic Injection Into the Corpus Callosum of Lewis Rats
  6. Basic Protocol 2: Cryopreparation of Lesion Sites for Electron Microscopy with High-Pressure Freezing
  7. Basic Protocol 3: Freeze Substitution of Brain Samples for Fixation and Embedding for Transmission Electron Microscopy
  8. Basic Protocol 4: Ultramicrotomy of the Injection Site
  9. Basic Protocol 5: Heavy Metal Staining of Thin Sections for Transmission Electron Microscopy
  10. Basic Protocol 6: Locating Injection Site by Electron Microscopy
  11. Reagents and Solutions
  12. Commentary
  13. Literature Cited

Assessment of the ultrastructure after intracerebral injection requires experience in electron microscopy to avoid mistaking artifacts for pathological structures. With the help of the stained semi-thin sections, the orientation of the tissue within the section can be determined. Depending on the time point of injection, the needle track can be used as a hallmark for early injections, whereas invasion of immune cells is an indicator for later lesions. It is crucial to investigate samples that were injected with vehicle only to be able to assess the tissue damage caused by the injection itself. Then, the treated samples can be imaged in such a way that the area caused by needle damage is not taken into account. Artifacts, such as freeze damage, need to be excluded from analysis (see Fig. 2.27.5).

Materials
  • Electron microscope
  • 1.

    Turn on the electron microscope in low magnification mode, identify the orientation of the tissue within the block by, e.g., using the trimmed corner as a landmark or the shape of the block.

    An image of the stained sections can be helpful to localize the injection site within the tissue and its approximate location.

  • 2.

    In the high magnification mode, observe an increased number of phagocytes (with an accumulation of debris) and the needle track, which indicate the localization of the injection site at high resolution (see Fig. 2.27.4).

figure

Figure 2.27.4. Localization of lesion sites in transmission electron microscopy. Common features of intracerebral injections are displayed in an overview electron micrograph in A, such as infiltrating immune cells (polymorphonuclear leucocytes, red arrow head), axonal swellings (blue arrow), and myelin damage (dashed, green arrow). The electron micrograph of a microglia in B shows engulfed myelin debris (white arrow head) and lipid droplets (dashed, brown arrow). Axonal swellings and myelin damage are shown in C and D, respectively. Scale bar for A, B: 2 µm, for C, D: 500 nm.

Reagents and Solutions

  1. Top of page
  2. Significance Statement
  3. Introduction
  4. Strategic Planning (Optional)
  5. Basic Protocol 1: Stereotactic Injection Into the Corpus Callosum of Lewis Rats
  6. Basic Protocol 2: Cryopreparation of Lesion Sites for Electron Microscopy with High-Pressure Freezing
  7. Basic Protocol 3: Freeze Substitution of Brain Samples for Fixation and Embedding for Transmission Electron Microscopy
  8. Basic Protocol 4: Ultramicrotomy of the Injection Site
  9. Basic Protocol 5: Heavy Metal Staining of Thin Sections for Transmission Electron Microscopy
  10. Basic Protocol 6: Locating Injection Site by Electron Microscopy
  11. Reagents and Solutions
  12. Commentary
  13. Literature Cited

Use deionized, distilled water in all recipes and protocol steps. For common stock solutions, see appendix 2A.

Anesthesia
  • 10% ketamine (Medistar, cat. no. 00615), store at 4ºC
  • 2% xylazine (Medistar, cat. no. 00914), store at 4ºC
  • Prepare final anesthesia mix: ketamine (60 mg/kg body weight) and xylazine (8 mg/kg body weight), prepare fresh
Epon resin
  • 21.4 g glycid ether 100 (Serva, cat. no. 21045.02)
  • 14.4 g 2-dodecenylsuccinic acid anhydride (DDSA) (Serva, cat. no. 20755.02)
  • 11.3 g methylnadic anhydride (MNA) (Serva, cat. no. 29452.03)

Stir solution for 10 min, then just before use, add 0.84 ml of 2,4,6-Tris(dimethyl-amino-methyl-)-phenol (DMP-30) (Roth, cat. no. 8621.1) and stir for an additional 20 min. Prepare fresh.

Richardson's methylene blue/azure II blue
  • 1% (w/v) azure II (Merck, cat. no. 109211) in ddH2O (stock solution I)
  • 1% (w/v) methylene blue (Merck, cat. no. 115943) in 1% sodium borate (stock solution II)

The stock solutions can be stored at room temperature for months. For staining of lipid-rich regions, equal volumes of the stock solutions are mixed and filtered directly before use.

Commentary

  1. Top of page
  2. Significance Statement
  3. Introduction
  4. Strategic Planning (Optional)
  5. Basic Protocol 1: Stereotactic Injection Into the Corpus Callosum of Lewis Rats
  6. Basic Protocol 2: Cryopreparation of Lesion Sites for Electron Microscopy with High-Pressure Freezing
  7. Basic Protocol 3: Freeze Substitution of Brain Samples for Fixation and Embedding for Transmission Electron Microscopy
  8. Basic Protocol 4: Ultramicrotomy of the Injection Site
  9. Basic Protocol 5: Heavy Metal Staining of Thin Sections for Transmission Electron Microscopy
  10. Basic Protocol 6: Locating Injection Site by Electron Microscopy
  11. Reagents and Solutions
  12. Commentary
  13. Literature Cited

Background Information

Electron microscopy is a very useful technique that provides information on the ultrastructure of a sample and is widely used to gain insight into the biology of cells and subcellular structures.

Conventional electron microscopy has advanced the understanding of physiology in various tissues. However, up until recently, tissue routinely needed to be fixed with aldehydes and afterwards dehydrated prior to embedding. State-of-the-art methods, such as cryopreparation by high-pressure freezing physically immobilize the sample by rapid freezing in liquid nitrogen and at ∼2048 bar (Moor and Riehle, 1968). This led to a vitrified sample due to the formation of amorphous ice (Dahl and Staehelin, 1989; Studer et al., 1989; Dubochet, 2007; Dubochet et al., 2007; Vanhecke et al., 2008), which is close to a native, fully hydrated structure (Korogod et al., 2015). The application of pressure reduces the increase of the sample volume and, therefore, reduces the ice crystal nucleation and its growth by lowering the freezing point (Moor, 1987; Steinbrecht and Müller, 1987; Dahl and Staehelin, 1989). Depending on the properties of the samples, the sample thickness is limited to <300 µm.

After cryofixation, the sample must be embedded into a plastic resin for sectioning at room temperature. The method presented here involves freeze substitution to remove the ice from the sample and replace it with organic solvents at temperatures below putative ice crystal growth. Simultaneously, the sample is kept at low temperatures (−90ºC) over a prolonged period of time. Therefore, the fixative can slowly and homogenously distribute within the sample while dehydrating the tissue at the same time (Steinbrecht and Müller, 1987). The actual fixation starts in situ when the samples are warmed up. This procedure prevents artifacts that are usually introduced upon fixation at room temperature (Schwarz and Humbel, 1989). For the freeze substitution cocktails, various protocols use uranyl acetate, osmium tetroxide, and/or glutaraldehyde in acetone, methanol, or ethanol. Acetone was found to replace water at a slower rate and yield better structural preservation (McDonald and Morphew, 1993). To enhance the membrane contrast, the stabilizing agent tannic acid (in acetone) (Akisaka and Shigenaga, 1983; Siksou et al., 2007) is applied as a mordant, whereas other protocols use potassium permanganate or combinations of glutaraldehyde, uranyl acetate, and osmium tetroxide (Ladinsky et al., 1999; McDonald and Müller-Reichert, 2002; Giddings, 2003), or the addition of water to the freeze substitution cocktail (Buser and Walther, 2008). For various tissues, a substitution mixture of osmium tetroxide and uranyl acetate in acetone achieved good results (Hess, 2007).

When assessing the effects of an injected substance on the ultrastructure (see Fig. 2.27.4), care must be taken so that freezing artifacts are not mistaken for pathology (see Fig. 2.27.5). Hence, a well-prepared sample is crucial for the morphological analysis of injections. Therefore, successful cryofixation and freeze substitution is a very helpful technique to assess the ultrastructure after intracerebral injection as it provides a close-to-native preservation of the tissue. Hence, subtle changes in the (sub)cellular structures of neurons and glia cells can be visualized prior to excessive damage.

figure

Figure 2.27.5. Freezing artifacts in cryopreparation for transmission electron microscopy. The electron micrographs indicate typical freezing artifacts, such as torn axons (see blue arrow in A and C), broken membranes (see dashed, green arrow in B), and badly preserved nuclei (see red arrowheads in C). Scale bar for A, B: 500 nm, for C: 2 µm.

Critical Parameters

The injections should be carried out very carefully and slowly to prevent excessive tissue damage. Therefore, the capillary should be moved very slowly and the injection mix should be infused very slowly. The needle should be left in place for 5 min for the agent to diffuse within the tissue and it should not be removed from the injection site too quickly.

During high-pressure freezing, the tissue should be processed fast and with minimal damage to the area that is processed. The handling of the tissue and its loading into the specimen carriers are the most critical steps within this protocol and should be practiced prior to performing an experiment.

After freezing samples, do not allow samples to warm up prior to freeze substitution as ice crystal formation causes freeze damage. Therefore, they should always be kept in liquid nitrogen and only be touched with liquid nitrogen–precooled forceps.

It is also crucial that the samples after freeze substitution are embedded completely flat. Otherwise, some parts of the tissue will be lost. Therefore, flatten the tissue by gently pressing down on the sample with forceps when placing the samples on Parafilm before covering them with Epon-filled gelatin capsules.

It is recommended to trim the edge of one corner of the Epon block (see Basic Protocol 4, step 6) to help quickly identify the orientation of the tissue within the section under a microscope.

Troubleshooting

See Table 2.27.2 for possible problems, causes, and solutions.

Table 2.27.2. Troubleshooting Guide
ProblemPossible causeSolution
Liquid is not injected in the brainClogged capillaryRemove the capillary, check for clogs (frequently blood clogs), cut the end of the capillary, or use a new capillary and repeat surgery
Excessive bleeding during injectionDisruption of blood vesselsTry avoiding blood vessels by trying new coordinates, ensure that the coordinates target the area of interest
Tissue damage after injectionToo fast injectionSlow down the injection speed and insert needle slowly
 Needle too big or cloggedUse new needle or thinner capillaries
Injection site not where expectedWrong stereotactic coordinatesRepeat, ensure that the coordinates target the area of interest
Injection site not visible upon cuttingMonastral blue too diluteUse a higher concentration of monastral blue
Many freezing artifactsTissue damage during high-pressure freezingRepeat, care must be taken when mounting sample into the carrier; adjust level of filler correctly to exclude air bubbles from sample carrier during sandwich assembly
  Check freezing rates and other parameters of the freezing cycle; these would indicate problems at the equipment level
Semi-thin section completely blueToo long incubation with Richardson's methylene blue/azure II blueReduce staining time
Sample over-contrastedToo long incubation during post-stainingReduce contrasting time
Structure of interest not contained within the sampleRegion of interest got lost during processingRepeat, make sure to correctly punch and trim sample

Statistical Analyses

The minimal requirement for statistical analyses of any effect of injected substances is at least three to five animals (n = 3 to 5). To assess effects at the cellular level for each animal, 25 fields of view at low magnification (7000×) covering 215 µm2 per field of view are imaged for cell counting. For subcellular assessment of effects, a higher magnification is required, e.g., 12,000× covering 17 µm2 per field of view. Then, after defining which categories are used to identify pathological changes (e.g., different stages of myelin disintegration as in Weil et al., 2016), the number of profiles falling into these categories is counted. Then, the distribution in the categories is calculated. If several categories are defined, statistical analysis is performed using one-way ANOVA.

Understanding Results

When assessing the effect of the administered substance on the ultrastructure of the tissue within the CNS, great care must be taken so as not to mistaken artifacts of high-pressure freezing (see Fig. 2.27.5) with anticipated results. Areas or profiles showing signs of freeze damage should be excluded from analysis. It is also crucial to always assess the vehicle-injected control first to avoid over-interpreting potential changes in the ultrastructure. Therefore, images should be taken some distance from the injection site, which suffers from tissue damage. Some examples of true pathological changes are axonal damage, myelin breakdown, and immune cell infiltration, shown in Figure 2.27.4.

Depending on the agents that were injected, different results can be obtained, contingent upon their effect on the different target structures. Therefore, knowledge of the effect of the substance is mandatory, such as correlation with histological results. For example, when antibodies against myelin proteins are administered, it can be anticipated that these have an effect on the ultrastructure of myelin. Time-course studies indicated the breakdown of the compact myelin lamellae into vesicular/tubular structures that could be correlated with a loss of myelin staining (Wrzos et al., 2014; Weil et al., 2016).

Time Considerations

Each intracerebral injection takes ∼1 hr per rat, whereas the cryopreparation of the tissue by high-pressure freezing takes ∼30 min per rat. The freeze substitution of up to 30 samples takes 6 days until the samples are embedded. Trimming and semi-thin sectioning of one sample takes ∼45 min to 1 hr per block. After staining semi-thin sections, the block can be furthered trimmed and ultrathin sections can be cut. These steps take 45 min to 1 hr per block. The samples are then contrasted with uranyl acetate for 20 min. The imaging takes between 1 and 3 hr per sample.

Acknowledgments

M.W. was funded by the Cluster of Excellence and DFG Research Center Nanoscale Microscopy and Molecular Physiology of the Brain and an ERC CoG Grant and SyNergy (M.S.).

Literature Cited

  1. Top of page
  2. Significance Statement
  3. Introduction
  4. Strategic Planning (Optional)
  5. Basic Protocol 1: Stereotactic Injection Into the Corpus Callosum of Lewis Rats
  6. Basic Protocol 2: Cryopreparation of Lesion Sites for Electron Microscopy with High-Pressure Freezing
  7. Basic Protocol 3: Freeze Substitution of Brain Samples for Fixation and Embedding for Transmission Electron Microscopy
  8. Basic Protocol 4: Ultramicrotomy of the Injection Site
  9. Basic Protocol 5: Heavy Metal Staining of Thin Sections for Transmission Electron Microscopy
  10. Basic Protocol 6: Locating Injection Site by Electron Microscopy
  11. Reagents and Solutions
  12. Commentary
  13. Literature Cited