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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
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
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