title: Neutral red as a probe for confocal laser scanning microscopy studies of plant roots.
abstract: OBJECTIVE
  Neutral red (NR), a lipophilic phenazine dye, has been widely used in various biological systems as a vital stain for bright-field microscopy. In its unprotonated form it penetrates the plasma membrane and tonoplast of viable plant cells, then due to protonation it becomes trapped in acidic compartments. The possible applications of NR for confocal laser scanning microscopy (CLSM) studies were examined in various aspects of plant root biology.
  
  
  METHODS
  NR was used as a fluorochrome for living roots of Phaseolus vulgaris, Allium cepa, A. porrum and Arabidopsis thaliana (wild-type and transgenic GFP-carrying lines). The tissues were visualized using CLSM. The effect of NR on the integrity of the cytoskeleton and the growth rate of arabidopsis primary roots was analysed to judge potential toxic effects of the dye.
  
  
  RESULTS
  The main advantages of the use of NR are related to the fact that NR rapidly penetrates root tissues, has affinity to suberin and lignin, and accumulates in the vacuoles. It is shown that NR is a suitable probe for visualization of proto- and metaxylem elements, Casparian bands in the endodermis, and vacuoles in cells of living roots. The actin cytoskeleton and the microtubule system of the cells, as well as the dynamics of root growth, remain unchanged after short-term application of NR, indicating a relatively low toxicity of this chemical. It was also found that NR is a useful probe for the observation of the internal structures of root nodules and of fungal hyphae in vesicular-arbuscular mycorrhizas.
  
  
  CONCLUSIONS
  Ease, low cost and absence of tissue processing make NR a useful probe for structural, developmental and vacuole-biogenetic studies of plant roots with CLSM.
authors.affiliation: Departamento de Biología Molecular de Plantas, Universidad Nacional Autónoma de México, Cuernavaca, Morelos, México. jdubrov@ibt.unam.mx
authors.forename: Joseph G
authors.initials: JG
authors.lastname: Dubrovsky
authors.forename: Martin
authors.initials: M
authors.lastname: Guttenberger
authors.forename: Andres
authors.initials: A
authors.lastname: Saralegui
authors.forename: Selene
authors.initials: S
authors.lastname: Napsucialy-Mendivil
authors.forename: Boris
authors.initials: B
authors.lastname: Voigt
authors.forename: Frantisek
authors.initials: F
authors.lastname: Baluska
authors.forename: Diedrik
authors.initials: D
authors.lastname: Menzel
mesh_terms: D018613:Microscopy, Confocal / Q000379:methods*; D015336:Molecular Probe Techniques; D038821:Mycorrhizae / Q000166:cytology; D009499:Neutral Red; D018517:Plant Roots / Q000033:anatomy & histology* / Q000254:growth & development; D014617:Vacuoles / Q000648:ultrastructure
chemical_list: D009499:Neutral Red