Abstract
There are various preparatory techniques for light microscopy permitting access to the inner structure of plant body and its development. Minute objects might be processed as whole-mount preparations, while voluminous ones should be separated into smaller pieces. Here we summarize some of the “classical” techniques to cut more voluminous objects into slices and access their inner structure either for simple anatomical analysis or for further processing (e.g., histochemistry, immunohistochemistry, in situ hybridization, enzyme histochemistry).
Key words
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Pearse AG (1980) Histochemistry (theoretical and applied): preparative and optical technology. Churchill Livingstone, Edinburgh
Pearse AG (1985) Histochemistry (theoretical and applied): analytical technology. Churchill Livingstone, Edinburgh
O’Brien TP, Mccully ME (1981) The study of plant structure: principles and selected methods. Termarcarphi Pty LTD, Melbourne
Ruzin SE (1999) Plant microtechnique and microscopy. Oxford University Press, Oxford
Fox CH, Johnson FB, Whiting J, Roller PP (1985) Formaldehyde fixation. J Histochem Cytochem 33:845–853
Medawar PB (1941) The rate of penetration of fixatives. J Royal Micro Soc 61:46–57
Bancroft JD, Gamble M (2008) Theory and practice of histological techniques. Churchill Livingstone, London
Mersey B, Mccully ME (1978) Monitoring of the course of fixation of plant cells. J Microsc 114:49–76
Coetzee J, van der Merwe CF (1985) Penetration rate of glutaraldehyde in various buffers into plant tissue and gelatin gels. J Microsc 137:129–136
Gardner RO (1975) An overview of botanical clearing technique. Biotech Histochem 50:99–105
Bybd DW Jr, Kirkpatrick T, Barker KR (1983) An improved technique for clearing and staining plant tissues for detection of nematodes. J Nematol 15:142–143
Stebbins GL Jr (1938) A bleaching and clearing method for plant tissues. Science 87:21–22
Malamy JE, Benfey PN (1997) Organization and cell differentiation in lateral roots of Arabidopsis thaliana. Development 124:33–44
Shobe WR, Lersten NR (1967) A technique for clearing and staining gymnosperm leaves. Bot Gaz 128:150–152
Sporne KR (1948) A note on a rapid clearing technique of wide application. New Phytol 47:290–291
Simpson JLS (1929) A short method of clearing plant tissues for anatomical studies. Biotech Histochem 4:131–132
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
Peterson CA, Fletcher RA (1973) Lactic acid clearing and fluorescent staining for demonstration of sieve tubes. Biotech Histochem 48:23–27
Lersten NR (1986) Modified clearing method to show sieve tubes in minor veins of leaves. Biotech Histochem 61:231–234
Herr JM Jr (1971) A new clearing-squash technique for the study of ovule development in angiosperms. Am J Bot 58:785–790
Beeckman T, Engler G (1994) An easy technique for the clearing of histochemically stained plant tissue. Plant Mol Biol Rep 12:37–42
Bougourd S, Marrison J, Haseloff J (2000) An aniline blue staining procedure for confocal microscopy and 3D imaging of normal and perturbed cellular phenotypes in mature Arabidopsis embryos. Plant J 24:543–550
Cunningham JL (1972) A miracle mounting fluid for permanent whole-mounts of microfungi. Mycologia 64:906–911
Truernit E, Bauby H, Dubreucq B, Grandjean O, Runions J et al (2008) High-resolution whole-mount imaging of three-dimensional tissue organization and gene expression enables the study of phloem development and structure in Arabidopsis. Plant Cell 20:1494–1503
Dubrovsky JG, Soukup A, Napsucialy-Mendivil S, Jeknic Z, Ivanchenko MG (2009) The lateral root initiation index: an integrative measure of primordium formation. Ann Bot 103:807–817
Kurihara D, Mizuta Y, Sato Y, Higashiyama T (2015) ClearSee: a rapid optical clearing reagent for whole-plant fluorescence imaging. Development 142(23):4168–4179
Ursache R, Andersen TG, Marhavý P, Geldner N (2018) A protocol for combining fluorescent proteins with histological stains for diverse cell wall components. Plant J 93(2):399–412
Zelko I, Lux A, Sterckeman T, Martinka M, Kollárová K et al (2012) An easy method for cutting and fluorescent staining of thin roots. Ann Bot 110:475–478
de Almeida Engler J, Van Montagu M, Engler G (1994) Hybridization in situ of whole-mount messenger RNA in plants. Plant Mol Biol Rep 12:321–331
Klebs E (1869) Die Einschmelzungs Methode, ein Beitrag zur mikroskopischen Technik. Arch Mikrosk Anat Entwicklungsmech 5:164–166
Johansen DA (1940) Plant microtechnique. McGraw-Hill Book Co. Inc, New York
Sass JE (1940) Elements of botanical microtechnique. McGraw-Hill Book Co Inc., New York, London
Vitha S, Baluska F, Jasik J, Volkmann D, Barlow PW (2000) Steedman’s wax for F-actin visualization. Dev Plant Soil Sci 89:619–636
Sartori N, Richter K, Dubochet J (1993) Vitrification depth can be increased more than 10-fold by high-pressure freezing. J Microsc 172:55–61
Quintana C (1994) Cryofixation, cryosubstitution, cryoembedding for ultrastructural, immunocytochemical and microanalytical studies. Micron 25:63–99
Beneš K (1973) On the media improving freeze-sectioning of plant material. Biol Plant 15:50–56
Tirichine L, Andrey P, Biot E, Maurin Y, Gaudin V (2009) 3D fluorescent in situ hybridization using Arabidopsis leaf cryosections and isolated nuclei. Plant Methods 5:11–18
Knapp E, Flores R, Scheiblin D, Scheiblin D, Modla S et al (2012) A cryohistological protocol for preparation of large plant tissue sections for screening intracellular fluorescent protein expression. Biotechniques 52:31–37
Zhang Z, Niu L, Chen X, Xu X, Ru Z (2012) Improvement of plant cryosection. Front Biol 7:374–377
Knox RB (1970) Freeze-sectioning of plant tissues. Biotech Histochem 45:265–272
Cocco C, Melis GV, Ferri GL (2003) Embedding media for cryomicrotomy: an applicativereappraisal. Appl Immunohistochem Mol Morphol 11:274–280
Williams DBG, Lawton M (2010) Drying of organic solvents: quantitative evaluation of the efficiency of several desiccants. J Org Chem 75:8351–8354
Pappas PW (1971) The use of a chrome alumgelatin (subbing) solution as a general adhesive for paraffin sections. Biotech Histochem 46:121–124
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
Ferri GL, Cocco C, Melis GV, Aste L (2002) Equipment testing and tuning: the cold-knife cryomicrotome microm HM-560. Appl Immunohistochem Mol Morphol 10:381–386
Barthel LK, Raymond PA (1990) Improved method for obtaining 3-microns cryosections for immunocytochemistry. J Histochem Cytochem 38:1383–1388
Soukup A, Tylová E (2014) Essential methods of plant sample preparation for light microscopy. Methods Mol Biol 1080:1–23
Acknowledgments
The current update of this chapter, originally published as ref. 47, has been supported by the NPUI-LO1417 project.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2019 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Soukup, A., Tylová, E. (2019). Essential Methods of Plant Sample Preparation for Light Microscopy. In: Cvrčková, F., Žárský, V. (eds) Plant Cell Morphogenesis. Methods in Molecular Biology, vol 1992. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9469-4_1
Download citation
DOI: https://doi.org/10.1007/978-1-4939-9469-4_1
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-4939-9468-7
Online ISBN: 978-1-4939-9469-4
eBook Packages: Springer Protocols