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Free Hand Sectioning and Staining With Toluidine Blue

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doi: 10.1093/aob/mci266. Epub 2005 Sep 28.

An improved method for clearing and staining free-hand sections and whole-mount samples

Affiliations

  • PMID: 16192293
  • PMCID: PMC4247103
  • DOI: 10.1093/aob/mci266

Free PMC article

An improved method for clearing and staining free-hand sections and whole-mount samples

Alexander Lux  et al. Ann Bot. 2005 Nov .

Free PMC article

Abstract

Background and aims: Free-hand sectioning of living plant tissues allows fast microscopic observation of internal structures. The aim of this study was to improve the quality of preparations from roots with suberized cell walls. A whole-mount procedure that enables visualization of exo- and endodermal cells along the root axis was also established.

Methods: Free-hand sections were cleared with lactic acid saturated with chloral hydrate, and observed with or without post-staining in toluidine blue O or aniline blue. Both white light and UV light were used for observation. Lactic acid was also used as a solvent for berberine, and fluorol yellow for clearing and staining the samples used for suberin observation. This procedure was also applied to whole-mount roots with suberized celllayers.

Key results: Clearing of sections results in good image quality to observe the tissue structure and cell walls compared with non-cleared sections. The use of lactic acid as a solvent for fluorol yellow proved superior to previously used solvents such as polyethylene glycol-glycerol. Clearing and fluorescence staining of thin roots such as those of Arabidopsis thaliana were successful for suberin visualization in endodermal cells within whole-mount roots. For thicker roots, such as those of maize, sorghum or tea, this procedure could be used for visualizing the exodermis in a longitudinal view. Clearing and staining of peeled maize root segments enabled observation of endodermal cell walls.

Conclusions: The clearing procedure using lactic acid improves the quality of images from free-hand sections and clearings. This method enhances the study of plant root anatomy, in particular the histological development and changes of cell walls, when used in combination with fluorescence microscopy.

Figures

F<sc>ig</sc>. 1.
Fig. 1.

Free-hand cross-sections of roots with and without clearing viewed with bright field or fluorescence microscopy. (A–F) Samples of aerial root of orchid (Oncidium sp.) in white light (A, C and E) and under UV light (B, D and F), without clearing (A and B), after clearing in lactic acid saturated with chloral hydrate (C and D) and with clearing as in (C) and (D), but after staining with toluidine blue (E and F). Note that the phloem poles are clearly visible under bright field after clearing and staining (C and E) and non-distinguishable in fresh sections (A). (G–I) Section of sorghum nodal root after clearing in lactic acid with chloral hydrate and staining with toluidine blue in white light (G), in UV light (H) and the overlapped image of the previous two pictures (I). Scale bars = 50 μm. Arrowheads indicate phloem poles; en, endodermis.

F<sc>ig</sc>. 2.
Fig. 2.

Free-hand cross-sections of roots with and without clearing, stained with berberine or fluorol yellow: (A–C) roots of onion; (D–H) adventitious roots of melon, (I) root of cucumber. (A, B) The section cleared in lactic acid with berberine, post-stained in an aqueous solution of berberine and with toluidine blue, in UV light (A) and overlapped micrographs in UV light and in white light (B). (C) The section cleared and stained with lactic acid containing berberine and post-stained with safranin, overlapped picture of photos in white light and UV light. (D–F) The same section cleared and stained with lactic acid containing fluorol yellow, in white light (D), in UV light (E) and the overlapped image of the two previous photos (F). Note the contrast of lamellar suberin stained with fluorol yellow and the regular, non-collapsed cells of endodermis. (G, H) The section after staining with fluorol yellow in polyethylene glycol–glycerol. Note irregular shape of partially collapsed endodermal cells. (I) Cucumber adventitious root cleared and stained with lactic acid containing fluorol yellow and post-stained with safranin. ep, epidermis; ex, exodermis; en, endodermis, mx, broad late metaxylem vessel. Scale bars: A = 100 μm (for A–C); D = 50 μm (for D–I).

F<sc>ig.</sc> 3.
Fig. 3.

(A–G) Longitudinal views of roots in fluorescence microscopy; (H–I) free-hand stem cross-section with clearing. (A, B) Surface view of maize seminal root after clearing in lactic acid with chloral hydrate, staining with aqueous solution of berberine and post-stained with safranin; focused on epidermal cells in white light; (B) the same area as in (A) but focused on exodermal cells in UV light. (C) Tea root after clearing and staining in lactic acid containing fluorol yellow, and post-staining with safranin in UV light. Red-stained epidermal cells are clearly visible (although partially out of focus) over the in-focus exodermal network depicted with bright yellow walls. (D–F) Root of arabidopsis after clearing and staining in lactic acid containing fluorol yellow in white light (D), in UV light (E) and overlapped picture after combining both previous images (F). Note the clear image of bright yellow-stained endodermis visible through the outer cortical and epidermal layers. (G) Endodermal network of sorghum nodal root in surface view in UV light. The sample was peeled (to remove the outer cortical layers and visualize the surface of the stele covered by endodermal cells), cleared and stained in lactic acid containing berberine. (H, I) Cross-section of Clematis stem after clearing in lactic acid saturated with chloral hydrate as overlapped images taken with white light and UV light. Scale bars: A = 50 μm (for A–G); H = 200 μm; I = 100 μm.

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Source: https://pubmed.ncbi.nlm.nih.gov/16192293/

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