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Double Labeling of Microtubules and Actin Filaments in Maize Leaf Division Zone

QN Qiong Nan
JM Janette Mendoza
MF Michelle Renee Facette

Published: Jun 20, 2019 DOI: 10.21769/BioProtoc.3262 Views: 4244

Edited by: Marisa Rosa

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Abstract

Maize leaves grow in a gradient. The base of young maize leaves contains a high proportion of symmetrically and asymmetrically dividing cells, making the leaf an effective model system for the study of cell division. Here, we describe a double staining method to visualize actin filaments and microtubules in fixed maize leaf tissues, using immunofluorescent staining for microtubules and fluorescent phalloidin staining for actin filaments. This method provides a technique for the study of the cytoskeleton during maize cell division.

Keywords: Maize search Actin filament search Microtubules search Double staining search Division zone search

Background

Immature growing maize leaves mature in a gradient. The developing leaf blade, which is distal to the pre-ligular band or ligule, has three primary developmental zones. Proximal to the base or ligule are undifferentiated cells undergoing symmetric divisions, followed by differentiating cells undergoing asymmetric divisions, and finally cells that are no longer dividing but undergoing rapid cell expansion (Sylvester et al., 1996; Facette et al., 2013). This makes the monocot leaf an excellent model for studying cell division and aspects of leaf development. The developmental progression observed in monocot leaves has been used to study photosynthetic and vascular bundle formation in maize (Li et al., 2010); stomatal divisions in Brachypodium distachyon (Raissig et al., 2016), rice (Wu et al., 2019) and maize (Facette et al., 2015); and cell division in maize (Hunter et al., 2012; Nelissen et al., 2012). Maize leaves have large cells excellent for imaging these processes. Live cell imaging of the immature leaf using fluorescent markers is powerful (Rasmussen, 2016), however this requires that plants express transgenic markers, which can be limiting. We present a whole-mount procedure that does not require difficult and time-consuming sectioning, to observe the actin and microtubule cytoskeleton in the developing maize leaf epidermis.

Materials and Reagents

  1. Glass Scintillation vial (20 ml)
  2. Parafilm
  3. Microscope slides 
  4. Microscope cover slips (use glass coverslips of appropriate thickness for your microscope objective, typically 1.5. Wide cover glasses are convenient, we recommend Fisher 12-544-D)
  5. Corning Netwell Inserts and 12-well plate (Corning, catalog number: 3478)
  6. M-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) (Thermo Fisher, catalog number: 22311)
  7. PIPES (Fisher Scientific, catalog number: 50-144-6210)
  8. HEPES (Alfa Aesar, catalog number: A1477709)
  9. MgSO4 (Fisher Scientific, catalog number: AC423900250)
  10. KOH (Fisher Scientific, catalog number: 18-605-601)
  11. Na2HPO4·7H2O (Fisher Scientific, catalog number: 18-606-699)
  12. NaCl (Fisher Scientific, catalog number: 18-606-422)
  13. KCl (Sigma-Aldrich, P5941)
  14. KH2PO4 (Fisher Scientific, catalog number: 18-602-144) 
  15. Methanol (Fisher Scientific, catalog number: 18-604-352)
  16. EGTA (Fisher Scientific, catalog number: O2783-100)
  17. 16% Formaldehyde (Pierce, catalog number: 28906)
  18. 10% Glutaraldehyde (Electron Microscopy Services, catalog number: 16120)
  19. Driselase powder (Sigma, catalog number: D8037)
  20. Pectolyase Y-23 powder (Duchefa Biochemie, catalog number: P8004)
  21. Anti-Tubulin antibody clone B-5-1-2 mouse (Sigma, catalog number: T5618)
  22. Goat anti-mouse AlexaFluor 568 (Invitrogen, catalog number: A11004)
  23. AlexaFluor 488-Phalloidin (Invitrogen, catalog number: A12379)
  24. Normal goat serum (NGS) (Thermo Fisher, catalog number: 16210)
  25. Triton X-100 (Sigma-Aldrich, catalog number: T8787)
  26. Dimethyl sulfoxide (DMSO)
  27. ProLong Diamond Antifade reagent, with or without DAPI (Invitrogen, with DAPI: P36962 or without DAPI P36961)
  28. MilliQ Water
  29. 30 mM MBS (stock solution) (see Recipes)
  30. Alexafluor 488-Phalloidin (stock solution) (see Recipes)
  31. Anti-Tubulin antibody clone B-5-1-2 mouse (freezer stock solution) (see Recipes)
  32. 2x PHEM buffer (see Recipes)
  33. 1x PHEM buffer (see Recipes)
  34. 10x PBS (see Recipes)
  35. 1x PBS (see Recipes)
  36. 10% Triton X-100 (see Recipes)
  37. Pre-fix solution (see Recipes)
  38. Fix solution (see Recipes)
  39. Enzyme solution (see Recipes)
  40. Extraction solution (see Recipes)
  41. Block solution (see Recipes)
  42. Staining solution I (see Recipes)
  43. Staining solution II (see Recipes)

Equipment

  1. Pipettes
  2. Scalpel with blade #11
  3. Fine tweezers (Electron Microscopy Services, catalog number: 78138-01)
  4. Tweezers
  5. Water spray bottle
  6. Orbital Shaker
  7. Vacuum chamber (Bel Art, F42025) connected to vacuum pump or building vacuum
  8. Speed Vacuum Concentrator
  9. Microscope system equipped for illumination for Alexafluor 488 (GFP) and Alexafluor 568 (RFP/Texas Red)
    Note: We recommend a point scanning confocal microscope, but other microscopes equipped with fluorescence are acceptable. Optional UV laser for DAPI if staining DNA using the ProLong Diamond Antifade with DAPI.

Procedure

Copyright: © 2019 The Authors; exclusive licensee Bio-protocol LLC.

Category

Plant Science > Plant cell biology > Cell structure

Developmental Biology > Morphogenesis > Cell structure

Cell Biology > Cell imaging > Fluorescence

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1 Q&A

When preparing the Fix solution, it is stated that AlexaFluor...
edit 1 Answer 2 Views Oct 30, 2020