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Toluidine Blue O Staining of Paraffin-Sectioned Maize Tissue

JS Josh Strable
JY Jeffrey R. Yen
MS Michael J. Scanlon
AS Anne W. Sylvester

Published: May 5, 2020 DOI: 10.21769/BioProtoc.3612 Views: 4540

Edited by: Marisa Rosa

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Abstract

Histology incorporates microtechnique, the preparation of living material for observation and microscopy to study the anatomy of cells, tissues and organs. Interpreting histological patterns in a specimen may help to correlate form and function at the cellular, tissue and organ levels. Many stain options and staining methods (“schedules”) for plant tissues have been described previously (Johansen, 1940; Jensen, 1962; O’Brien and McCully, 1981; Sylvester and Ruzin, 1994; Ruzin, 1999). Here, we present a straightforward, inexpensive protocol that uses Toluidine Blue O (TBO) to stain maize tissue. TBO is a general metachromatic stain that can be used on paraffin-embedded histological sections, as well as fixed, hand-sectioned material. The value of TBO as a stain is the inherent metachromasia, whereby the dye is altered by pH resulting in violet blue color variants. This property is particularly useful even in sectioned material for identifying acidic components of plant cells. We demonstrate the utility of TBO staining for cellular-, tissue- and organ-level studies using maize seedling root, shoot apex and mature leaf (Figure 2).

Keywords: Maize search Histology search Toluidine Blue O search Paraffin-section search Deparaffinized-staining search

Background

Materials and Reagents

  1. Consumables
    1. Paper towels
    2. Kimwipes
    3. Razor blades (single edge, steel)
    4. Pencil
    5. Parafilm
    6. Adhesive Glass Microscope Slides (VWR, VistaVisionTM HistoBond®, catalog number: 16004-406 )
    7. Cover slips (25 x 60 mm; VWR, catalog number: 89082-272 )
    8. Glass Pasteur pipettes
    9. Glass vials with caps (VWR, catalog number: 66012-022 )
    10. Paraplast Plus® (McCormick Scientific, catalog number: 15159-464 )
    11. Cups with lids (VWR, catalog number: 89508-714 )
    12. Biopsy cassettes (VWR, catalog number: 25608-756 )
    13. Disposable base molds (VWR, catalog number: 100501-996 )
    14. Embedding ring (VWR, catalog number: 87002-374 )

  2. Biological material
    Maize tissue (e.g., shoot apex, stem, fully expanded leaf blade or root)

  3. Reagents
    1. Distilled water
    2. Ethanol (VWR, catalog number: EM-EX0276-3S )
    3. Histo-Clear II (VWR, catalog number: 101412-882 )
    4. Acetic acid glacial (VWR, catalog number: BDH3094-2.5LG )
    5. 37% Formaldehyde solution (VWR, catalog number: 97064-604 )
    6. Sodium borate (VWR, catalog number: 470302-454 )
    7. Toluidine Blue O (VWR, catalog number: 97062-168 )
    8. Permount (Fisher, catalog number: SP15-100 )
    9. Ethanol series: dilutions to 95%, 85%, 70%, 50% in distilled water
    10. Eosin Y (VWR, catalog number: 97061-034 )
    11. FAA fixative (100 ml) (see Recipes)

Equipment

  1. Forceps (VWR, catalog number: 82027-386 )
  2. Slide warmer (set to 37-42 °C)
  3. Oven set at 42 °C
  4. Vacuum pump
  5. Microtome
  6. Fine paintbrushes (e.g., Virtuous Arts Fine Paintbrushes, amazon.com)
  7. Stainless steel slide rack with handle (VWR, catalog number: 25461-014 )
  8. Tupperware (e.g., medium rectangular 15.5 x 8.1 x 7.3 cm of volume 412 ml)
  9. Glass staining dish (VWR, catalog number: 25461-016 ) with cover (VWR, catalog number: 25461-018 )
  10. Orbital shaker
  11. Microscope equipped with camera

Procedure

Copyright: © 2020 The Authors; exclusive licensee Bio-protocol LLC.

Category

Plant Science > Plant developmental biology > Morphogenesis

Plant Science > Plant cell biology > Tissue analysis

Cell Biology > Tissue analysis > Tissue imaging

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