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Brain Tissue Preparation, Sectioning, and Staining

  • Jingdong ZhangEmail author
  • Huangui Xiong
Protocol
Part of the Springer Protocols Handbooks book series (SPH)

Abstract

This chapter will summarize commonly used methods of brain tissue preparation, sectioning, and staining. History, development, and novel application of those methods in neuroscience will be presented herein so students can fully grasp the topic of brain tissue preparation, sectioning, and staining. The selectively introduced methods in this chapter are as follows: (1) Transcardial perfusion, especially on rats and mice, with a supplemental video referenced online to show perfusion procedure for adult rats. (2) Cryoprotection and frozen section cutting, with emphasis on the importance of cryoprotection for obtaining superior histological staining. Examples will be based on the author’s own experiences. (3) Paraffin embedding and sectioning, in which a general procedure is introduced. (4) Vibratome sectioning and application in histochemical staining for electron microscopy (EM) studies. In this chapter, procedures introduced will be based on cutting 50 μm sections for EM histochemical stains and plate embedding. We described detailed protocols for tissue treatment using freeze and thaw and ABC kit to reveal immunostain, two key steps in all immuno-EM. (5) Following histochemical staining, the stained structures need to be further observed under EM. These ultrathin sections are generally 1.5 mm long and 0.5 mm wide. To overcome the obstacle of extracting such a small area while ensuring it contains the desired labeling, a plate embedding method is used, which is described in this chapter. We will also illustrate a protocol for production of self-made siliconized slides used for plate embedding. (6) A protocol for a well-known traditional neural stain, Golgi’s stain, is presented here, with representative microimages. This section also highlights examples of new applications of this long-established method with Golgi’s stain kit in modern neuroscience research with representative images. (7) Protocols for Nissl staining, another commonly used neuron stain, and relevant photos. Two Nissl stains detailed here are cresyl violet and neutral red stains. (8) The methodology of hematoxylin and eosin (HE) stain, a broadly used histology stain method in both research and clinical labs, will be discussed here with detailed protocol. (9) Finally, we introduce a myelin sheath stain, Luxol fast blue, with practicable protocol. Due to demyelination being a highly researched area, this method is very applicable in current day studies of demyelinating diseases and animal models thereof.

Keywords

Brain tissue preparation Brain tissue sectioning Brain tissue staining Classical neuron staining Electron microscopy preparation 

References

  1. Bjorklund A (1983) Fluorescence histochemistry of biogenic monoamines. In: Bjoklund A, Hokfelt T (eds) Handbook of neuroanatomy. Elsevier, Amsterdam, pp 50–121Google Scholar
  2. Carriel V, Garzon I, Alaminos M, Campos A (2011) Evaluation of myelin sheath and collagen reorganization pattern in a model of peripheral nerve regeneration using an integrated histochemical approach. Histochem Cell Biol 136:709–717CrossRefPubMedGoogle Scholar
  3. Gage GJ, Kipke DR, Shain W (2012) Whole animal perfusion fixation for rodents. J Vis Exp (65)pii:3564Google Scholar
  4. Hasegawa R, Takami S, Nishiyama F (2008) Immunoelectron microscopic analysis of the distribution of tyrosine kinase receptor B in olfactory axons. Anat Sci Int 83:186–194CrossRefPubMedGoogle Scholar
  5. Jones EG (2010) Cajal’s debt to Golgi. Brain Res Rev 66:83–91CrossRefPubMedGoogle Scholar
  6. Kluver H, Barrera E (1953) A method for the combined staining of cells and fibers in the nervous system. J Neuropathol Exp Neurol 12:400–403CrossRefPubMedGoogle Scholar
  7. Li JL, Wang D, Kaneko T, Shigemoto R, Nomura S, Mizuno N (2000) The relationship between neurokinin-1 receptor and substance P in the medullary dorsal horn: a light and electron microscopic immunohistochemical study in the rat. Neurosci Res 36:327–334CrossRefPubMedGoogle Scholar
  8. Li L, Yun SH, Keblesh J, Trommer BL, Xiong H, Radulovic J, Tourtellotte WG (2007) Egr3, a synaptic activity regulated transcription factor that is essential for learning and memory. Mol Cell Neurosci 35:76–88CrossRefPubMedPubMedCentralGoogle Scholar
  9. Luo P, Haines A, Dessem D (2001) Elucidation of neuronal circuitry: protocol(s) combining intracellular labeling, neuroanatomical tracing and immunocytochemical methodologies. Brain Res Brain Res Protoc 7:222–234CrossRefPubMedGoogle Scholar
  10. Pinaud R, Jeong JK (2010) Duplex in situ hybridization in the study of gene co-regulation in the vertebrate brain. Methods Mol Biol 611:115–129CrossRefPubMedGoogle Scholar
  11. Pinaud R, Mello CV, Velho TA, Wynne RD, Tremere LA (2008) Detection of two mRNA species at single-cell resolution by double-fluorescence in situ hybridization. Nat Protoc 3:1370–1379CrossRefPubMedGoogle Scholar
  12. Pistorio AL, Hendry SH, Wang X (2006) A modified technique for high-resolution staining of myelin. J Neurosci Methods 153:135–146CrossRefPubMedGoogle Scholar
  13. Rocchietta (1968) Franz Nissl (1860–1919), neuropathologist. JAMA 205:460–461Google Scholar
  14. Soontornniyomkij V, Choi C, Pomakian J, Vinters HV (2010) High-definition characterization of cerebral beta-amyloid angiopathy in Alzheimer’s disease. Hum Pathol 41:1601–1608CrossRefPubMedPubMedCentralGoogle Scholar
  15. Steencken AC, Siebert JR, Stelzner DJ (2009) Lack of axonal sprouting of spared propriospinal fibers caudal to spinal contusion injury is attributed to chronic axonopathy. J Neurotrauma 26:2279–2297CrossRefPubMedGoogle Scholar
  16. Tang ZY, Shu B, Cui XJ, Zhou CJ, Shi Q, Holz J, Wang YJ (2009) Changes of cervical dorsal root ganglia induced by compression injury and decompression procedure: a novel rat model of cervical radiculoneuropathy. J Neurotrauma 26:289–295CrossRefPubMedGoogle Scholar
  17. Triarhou LC, Del Cerro M (2012) Ramon y Cajal erroneously identified as Camillo Golgi on a souvenir postage stamp. J Hist Neurosci 21:132–138CrossRefPubMedGoogle Scholar
  18. Wohlrab F, Henoch U (1988) The life and work of Carl Weigert (1845–1904) in Leipzig 1878–1885. Zentralbl Allg Pathol 134:743–751PubMedGoogle Scholar
  19. Yabuta NH, Yasuda K, Nagase Y, Yoshida A, Fukunishi Y, Shigenaga Y (1996) Light microscopic observations of the contacts made between two spindle afferent types and alpha-motoneurons in the cat trigeminal motor nucleus. J Comp Neurol 374:436–450CrossRefPubMedGoogle Scholar
  20. Yoshida A, Mukai N, Moritani M, Nagase Y, Hirose Y, Honma S, Fukami H, Takagi K, Matsuya T, Shigenaga Y (1999) Physiologic and morphologic properties of motoneurons and spindle afferents innervating the temporal muscle in the cat. J Comp Neurol 406:29–50CrossRefPubMedGoogle Scholar
  21. Zhang J, Pendlebury WW, Luo P (2003) Synaptic organization of monosynaptic connections from mesencephalic trigeminal nucleus neurons to hypoglossal motoneurons in the rat. Synapse 49:157–169CrossRefPubMedGoogle Scholar
  22. Zhang J, Luo P, Ro JY, Xiong H (2012) Jaw muscle spindle afferents coordinate multiple orofacial motoneurons via common premotor neurons in rats: an electrophysiological and anatomical study. Brain Res 1489:37–47CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  1. 1.Department of Pharmacology and Experimental NeuroscienceUniversity of Nebraska Medical Center, Durham Research CenterOmahaUSA

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