Fixation and Immunolabeling of Brain Slices: SNAPSHOT Method

Lasse Dissing‐Olesen1, Brian A. MacVicar1

1 University of British Columbia, Vancouver, British Columbia
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 1.23
DOI:  10.1002/0471142301.ns0123s71
Online Posting Date:  April, 2015
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Abstract

Acute brain slices are widely used in neuroscience because this preparation enables pharmacological interventions in a timely manner, similar to what is currently done in cultured cell studies, while preserving the natural cytoarchitecture. However, compared with cells in culture and thin cryostat sections, acute brain slices are not commonly used for immunolabeling because of poor fixation and antibody penetration. Thus, we have established a novel protocol to overcome these issues. We named this protocol SNAPSHOT (StaiNing of dynAmic ProcesseS in HOt‐fixed Tissue) because it describes a simple approach for preserving the morphology of fine dynamic cellular processes at the exact time of fixation and for improving the penetration of antibodies. We have previously shown that SNAPSHOT preserves the ultrastructure of the tissue and allows for a uniform immunolabeling throughout a 300 μm thick slice. SNAPSHOT has recently proven to be beneficial in addressing several unique biological questions. © 2015 by John Wiley & Sons, Inc.

Keywords: fixation; immunohistochemistry; acute brain slices; microglia

     
 
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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1:

  Materials
  • Phosphate‐buffered paraformaldehyde (PFA; FD Neurotechnologies, cat. no. PF101 tablets; Oxoid, cat. no. BR0014G)
  • Brain slices (see Dissing‐Olesen et al., )
  • Artificial cerebrospinal fluid (ACSF; see recipe)
  • 0.1 M PBS (tablets; Oxoid, cat. no. BR0014G): prepare according to the manufacturer's directions
  • Dimethyl sulfoxide (DMSO; Fisher Chemicals, D128‐1)
  • 2% (v/v) Triton X‐100 (J.T. Baker, cat. no. X198‐05)
  • Permeabilizing/washing solution (see recipe)
  • Blocking solution: add 10% (v/v) serum from the species in which the secondary (or tertiary, if required) antibody is raised to permeabilizing/washing solution (see recipe); prepare fresh
  • Primary antibodies appropriate to the target being studied
  • Secondary antibodies appropriate to the primary antibodies being used
  • Staining solution: added 2.5% (v/v) serum from the same species as used for blocking to permeabilizing/washing solution (see recipe); prepare fresh
  • Krazy Glue (Elmer's)
  • Corn oil (Sigma, cat.no. C8267)
  • Water bath (e.g., VWR, cat. no. 89501‐460)
  • Fume hood (e.g., Bedcolab)
  • 6‐ and 12‐well tissue culture plates (Falcon, cat. no. 08‐772‐1B and cat. no. 08‐772‐29, respectively)
  • 15.6‐cm transfer pipets (Electron Microscope Sciences, cat. no. 70960‐1)
  • Metal spatula (VWR, cat. no. 82027‐528), with tip bent 90°
  • Platform rotator (e.g., Fisher Scientific, cat. no. 13‐687‐705PM) or 360° rotisserie (e.g., Fisher Scientific)
  • 6 in. × 8 in. Reloc Zippit 4 mil reclosable plastic bags (US Plastic, cat. no, 48367)
  • Manual impulse sealer (e.g., Tecknopack, model E‐MMS‐200)
  • 1.7‐ml microcentrifuge tube (VWR, cat. no. 20172‐778)
  • Microscope slides (Fisher Scientific, cat. no. 12‐544‐2)
  • Cover glasses (Fisher Scientific, cat. no. 12‐548‐5 G)
  • Two‐photon scanning microscope (e.g., Coherent Chameleon Ultra II laser coupled to a Zeiss LSM7MP‐AX10 microscope with a Zeiss 20 × W/1.0 NA objective)
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Figures

Videos

Literature Cited

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Key References
  Dissing‐Olesen, L., LeDue, J.M., Rungta, R.L., Hefendehl, J.K., Choi, H.B., and MacVicar, B.A. 2014. See above.
  Mills, F., Bartlett, T.E., Dissing‐Olesen, L., Wisniewska, M.B., Kuznicki, J., Macvicar, B.A., Wang, Y.T., and Bamji, S.X. 2014. See above.
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