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Effects of fixatives and permeabilisation buffers on pollen tubes: implications for localisation of actin microfilaments using phalloidin staining

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Summary

Standard methods for visualising microfilament (MF) arrays in pollen tubes using rhodamine phalloidin (RP) involve treatment of living tubes with a variety of stabilising, permeabilising, and fixation agents. Video differential interference contrast (DIC) microscopy has been used to investigate the effect of these agents on normalNicotiana pollen tube structure and activity. Most of these agents were found to induce extensive axial translocations generally starting with tipward contractions. These movements were less extensive in the apex compared to more distal regions, however, tips often suffered swelling damage. RP staining patterns of the actin cytoskeleton were highly variable within fixation treatments. In addition to investigating agents used by other authors on pollen tubes, we investigated the efficacy of pretreatment withm-maleimido-benzyol N-hydroxysuccinimide ester (MBS). This resulted in less disruption to the pollen tubes, especially when used alone in growth medium. It also gave better HP-labelling than that achieved in standard aldehyde-fixed specimens. We conclude that standard preparation methods do not faithfully preserve in vivo cytoplasmic integrity in pollen tubes so that subsequent images of MF distribution may be misleading.

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Abbreviations

GM:

growth medium

RP:

rhodamine phalloidin

MBS:

m-maleimidobenzoyl N-hydroxysuccinimide ester

DIC:

differential interference contrast

MF:

microfilament

PIPES:

piperazine-N, N-bis-2-ethanesulphonic acid

PME:

50 mM PIPES buffer, pH 6.8, amended with 2.0 mM EGTA and 1 mM MgSO4

EB:

extraction buffer

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Authors and Affiliations

  1. Botany Department, University College Dublin, Belfield, Dublin 4, Ireland

    Fiona P. Doris & Martin W. Steer

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  1. Fiona P. Doris
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  2. Martin W. Steer
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Correspondence to Martin W. Steer.

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Dedicated to Professor Eldon H. Newcomb in recognition of his contributions to cell biology

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Doris, F.P., Steer, M.W. Effects of fixatives and permeabilisation buffers on pollen tubes: implications for localisation of actin microfilaments using phalloidin staining. Protoplasma 195, 25–36 (1996). https://doi.org/10.1007/BF01279184

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  • DOI: https://doi.org/10.1007/BF01279184

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