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Immunolabeled microtubules and microfilaments are visible in multiple cell layers of rye root tip sections

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Summary

A protocol was developed to observe plant microtubules and actin microfilaments in large tissue samples without physical sectioning. Rye (Secale cereale L. cv. Rymin) root tip pieces from two-day-old seedlings were fixed and processed for immunolabeling. Incubation times of 24–48 h were required to insure adequate penetration of fixatives, antibodies, and washing buffers. Clearing of the tissue with methyl salicylate reduced background auto-fluorescence that would otherwise interfere with the resolution of cytoskeletal structures. Microtubules or microfilaments in 5–7 cell layers were visualized using the optical-sectioning capability of laser scanning confocal microscopy (LSCM) and projected as three-dimensional images. The three-dimensional character of the cytoskeletal elements is retained when viewing stained cells of intact tissue. The net-like character of a microfilament array radiating out from a single point into the cytoplasm is maintained when the cells are stained in intact root tip pieces and imaging is accomplished in situ.

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Abbreviations

Cy3:

cyanine 3.18-conjugated goat anti-mouse IgG

FITC-M:

fluorescein isothiocyanate conjugated anti-mouse IgG

IFB:

immunofluorescence buffer

LSCM:

laser scanning confoeal microscopy

TPBS:

phosphate-buffered saline with 0.1% Triton X-100

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Authors and Affiliations

  1. Department of Horticultural Science, University of Minnesota, 55108, St. Paul, MN, USA

    M. E. Ericson & J. V. Carter

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  1. M. E. Ericson
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  2. J. V. Carter
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Ericson, M.E., Carter, J.V. Immunolabeled microtubules and microfilaments are visible in multiple cell layers of rye root tip sections. Protoplasma 191, 215–219 (1996). https://doi.org/10.1007/BF01281819

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  • DOI: https://doi.org/10.1007/BF01281819

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