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Preparing Immunolocalization Slides of Maize Meiotic Chromosomes for Three-dimensional Microscopy

QD Qian Du
WP Wojciech P. Pawlowski

Published: Jul 20, 2020 DOI: 10.21769/BioProtoc.3691 Views: 1907

Edited by: Marisa Rosa

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Abstract

The protocol provides fully detailed steps for preparing microscopic slides of acrylamide-embedded maize meiotic cells. This method is particularly useful for examining chromatin structure and chromosome arrangement without destroying the three-dimensional organization of the nucleus.

Keywords: Three-dimensional microscopy search Immunolocalization search Meiocytes search Chromosomes search Maize search

Background

Meiosis is a dynamic process involving homologous chromosomes pairing, synapsis, and recombination. Cytological analysis to investigate localization and dynamics of meiotic proteins are crucial for understanding details of these processes. In many microscopic slide preparation methods, spatial chromatin organization is damaged by either mechanical processing or chemical solvents. Here, we describe a three-dimensional microscopy protocol to analyze chromatin structure and meiotic protein localization without disturbing nuclear organization.

Materials and Reagents

  1. 15-ml Falcon tubes (VWR, catalog number: 62406-200 )
  2. 1.5-ml microcentrifuge tubes (Fisher Scientific, catalog number: 05-408-129 )
  3. Small Petri dishes (VWR, catalog number: 25384-302 )
  4. Microscopic glass slides (Thermo Fisher Scientific, catalog number: 12-550D )
  5. Scalpels (Fine Science Tools, catalog numbers: 10003-12 and 10010-00 )
  6. Parafilm (Bemis, catalog number: PM-999 )
  7. Glass microscope coverslip (VWR, catalog number: 48366-067 )
  8. Six-well tissue culture plate (Becton Dickinson, catalog number: 35-1146 )
  9. Tupperware box
  10. Diamond marker pen (VWR, catalog number: 103302-470 )
  11. Nail polish
  12. BSA (bovine serum albumin, Sigma, catalog number: A9418 )
  13. 16% paraformaldehyde (Electron Microscopy Sciences, catalog number: 15710 )
  14. Acrylamide/bisacrylamide solution (30% acrylamide + 0.8% bisacrylamide; Roche, catalog number: 11-685-821-001 )
  15. PIPES (Sigma-Aldrich, catalog number: P6757 )
  16. KCl (Sigma-Aldrich, catalog number: P3911 )
  17. NaCl (Sigma-Aldrich, catalog number: S7653 )
  18. EDTA (Fisher, catalog number: AC118430010 )
  19. EGTA (OmniPur, catalog number: 4100-50G )
  20. Na2HPO4 (Fisher, catalog number: BP332-500 )
  21. KH2PO4 (Fisher, catalog number: P380-500 )
  22. Triton X-100 (Sigma-Aldrich, catalog number: T8787 )
  23. Tween 20 (Fisher, catalog number: BP337-100 )
  24. DABCO (1,4 diazabicyclo-[2,2,2] octane, Sigma, catalog number: D-2522 )
  25. Tris (Fisher, catalog number: BP152-500 )
  26. Glycerol (Fisher, catalog number: BP229-1 )
  27. DAPI solution (4,6-diamidino-2-phenylindole; Vector Laboratories, catalog number: H-1200 )
  28. Spermine tetra HCl (Sigma, catalog number: S-2876 )
  29. Spermidine (Calbiochem, catalog number: 56766 )
  30. DTT (Calbiochem, catalog number: 233155 )
  31. Sorbitol (Sigma, catalog number: S1876 )
  32. Ammonium persulfate (Acros, catalog number: 327081000 )
  33. Sodium sulfite (Fisher, catalog number: S447-500 )
  34. 1x Bufffer A (see Recipes)
  35. 2x Buffer A (see Recipes)
  36. 10x Buffer A salts (see Recipes)
  37. Spermine stock (see Recipes)
  38. Spermidine stock (see Recipes)
  39. DTT stock (see Recipes)
  40. Sorbitol solution (see Recipes)
  41. 20% (w/v) Sodium sulfite (see Recipes)
  42. 20% (w/v) Ammonium persulfate (see Recipes)
  43. 10x PBS (see Recipes)
  44. Permeabilization buffer (see Recipes)
  45. Blocking buffer (see Recipes)
  46. Washing buffer (see Recipes)
  47. DABCO antifade solution (see Recipes)
  48. DAPI solution (see Recipes)
  49. Carnoy’s fixative (see Recipes)

Equipment

  1. Fine-tip forceps (Fine Science Tools, catalog number: 11252-30 )
  2. -20 °C freezer
  3. Dissecting microscope
  4. Fluorescence microscope
  5. Rocking platform shaker

Procedure

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

Category

Plant Science > Plant cell biology > Cell imaging

Cell Biology > Cell imaging > Fixed-tissue imaging

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