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Improved Methods for Clearing and Staining of Plant Samples


The free-hand sectioning represents a suitable method for fast microscopic observation and microphotography. The aim of this protocol is to improve the quality of preparations with emphasis on roots and visualization of suberized and lignified cell walls. Free-hand sections were cleared with lactic acid or lactic acid saturated with chloral hydrate, and observed with or without a post-staining step in toluidine blue O or aniline blue. For suberin detection in fluorescence microscopy, lactic acid was used as a solvent for berberine and fluorol yellow. This method was applied to both hand sections and whole-mount thin roots with suberized cell layers. Cleared samples were also exposed to phloroglucinol and HCl (hydrochloric acid) for detection of lignin. Clearing provides superior image quality for observation compared to non-cleared samples.


  • Casparian bands
  • Clearing
  • Endodermis
  • Exodermis
  • Fluorescence microscopy
  • Hand sections
  • Lignin
  • Suberin
  • Whole-mount samples

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  • DOI: 10.1007/978-3-319-19944-3_10
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  1. Žárský V, Cvrčková F (eds) (2014) Plant cell morphogenesis, vol 1080. Springer, New York (Methods Mol Biol)

    Google Scholar 

  2. Herr JM Jr (1993) Clearing techniques for the study of vascular plant tissues in whole structures and thick sections. In: Golman CA, Hauta PL, O`Donnell MA, Andrews SE, van der Heiden R (eds) Tested studies for laboratory teaching, vol 5. Proceedings of the 5th Workshop/Conference of the Association for Biology Laboratory Education (ABLE), pp 63–84

    Google Scholar 

  3. Herr JM Jr (1971) A new clearing-squash technique for the investigation of vascular plants morphology. Am J Bot 58:785–790

    CrossRef  Google Scholar 

  4. Herr JM Jr (1974) A clearing squash technique for the study of the ovule and megagametophyte development in angiosperms. In: Radford AE, Dickinson C, Massey JR, Bell CR (eds) Vascular plant systematics. Harper and Row, New York, pp 230–235

    Google Scholar 

  5. O`Brien TP (1974) Autoclaving as an aid in the clearing of plant specimens. Stain Tech 49:175–176

    CAS  Google Scholar 

  6. Kurth E (1978) A modified method for clearing leaves. Stain Tech 53:291–293

    CAS  Google Scholar 

  7. Stebbins GL Jr (1938) A bleaching and clearing method for plant tissues. Science 87:21–22

    CrossRef  PubMed  Google Scholar 

  8. Peterson CA, Fletcher RA (1973) Lactic acid clearing and fluorescent staining for demonstration of sieve tubes in minor veins of leaves. Biotech Histochem 48:23–27

    CAS  CrossRef  Google Scholar 

  9. Beeckman T, Engler G (1994) An easy technique for the clearing of histochemically stained plant tissue. Plant Mol Biol Rep 12:37–42

    CrossRef  Google Scholar 

  10. Lux A, Morita S, Abe J, Ito K (2005) An improved method for clearing and staining free-hand sections and whole-mount samples. Ann Bot 96:989–996

    PubMed Central  CrossRef  PubMed  Google Scholar 

  11. Villani TS, Koroch AR, Simon JE (2013) An improved clearing and mounting solution to replace chloral hydrate in microscopic applications. Applic Plant Sci 1:1300016

    Google Scholar 

  12. Eo J-K (2012) A simple technique for cross-sectioning Gymnosperm needle leaves using microtome. Turk J Bot 36:213–216

    Google Scholar 

  13. Dubrovsky JG, Soukup A, Napsucialy-Mendivil S, Jeknić Z, Ivanchenko MG (2009) The lateral root initiation index: an integrative measure of primordium formation. Ann Bot 103:807–817

    PubMed Central  CAS  CrossRef  PubMed  Google Scholar 

  14. Soukup A, Tylová E (2014) Essential methods of plant sample preparation for light microscopy. In: Žárský V, Cvrčková F (eds) Plant cell morphogenesis. Methods in molecular biology, vol 1080. Springer, New York, pp 1–23

    CrossRef  Google Scholar 

  15. Brundrett MC, Enstone DE, Peterson CA (1988) A berberine-aniline blue fluorescent staining procedure for suberin, lignin, and callose in plant tissue. Protoplasma 146:133–142

    CrossRef  Google Scholar 

  16. Brundrett MC, Kendrick B, Peterson CA (1991) Efficient lipid staining in plant material with Sudan red 7B or Fluorol yellow 088 in polyethylene glycol-glycerol. Biotech Histochem 66:111–116

    CAS  CrossRef  PubMed  Google Scholar 

  17. Zelko I, Lux A, Sterckeman T, Martinka M, Kollárová K, Lišková D (2012) An easy method for cutting and fluorescent staining of thin roots. Ann Bot 110:475–478

    PubMed Central  CAS  CrossRef  PubMed  Google Scholar 

  18. Kisser J, Steininger A (1950) Versuche zur Haltbarmachung der Phloroglucin–Salzsäure–Reaktion. Mikroskopie 5:39–41

    Google Scholar 

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The work was supported by Slovak Research and Development Agency under the contract Nr. APVV-0140-10, APVV SK-CN-0016-12, APVV SK-FR-2013-0029, VEGA 1/0817/12 and was a part of COST FA 0905 Action.

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Correspondence to Alexander Lux .

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Lux, A., Vaculík, M., Kováč, J. (2015). Improved Methods for Clearing and Staining of Plant Samples. In: Yeung, E., Stasolla, C., Sumner, M., Huang, B. (eds) Plant Microtechniques and Protocols. Springer, Cham.

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