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Immunolabeling of Maize Meiocytes

Mateusz Zelkowski Mateusz Zelkowski
WP Wojciech P. Pawlowski

Published: Apr 5, 2020 DOI: 10.21769/BioProtoc.3567 Views: 3204

Edited by: Marisa Rosa

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Abstract

This protocol describes a quick method for immunolocalization of meiotic proteins in maize. It includes a fixation step that allows for long-term storage of material and provides good preservation of chromatin structure.

Keywords: Immunolocalization search Meiosis search Meiocytes search Chromosomes search Maize search

Background

Meiocytes are generative cells, in which specialized processes of homologous chromosomes pairing, synapsis, and recombination take place, followed by chromosome segregation to create haploid nuclei. Elucidating localization of meiotic proteins is crucial for understanding these processes. Although many variations of this protocol are possible, we found that the microwave heating step is critical for it to work reliably for immunolocalization in maize.

Materials and Reagents

  1. 15-ml Falcon tubes (VWR, catalog number: 62406-200 )
  2. 22 x 22 mm glass coverslips (VWR, catalog number: 48366-227 )
  3. Parafilm (Bemis, catalog number: PM-999 )
  4. Small Petri dishes (VWR, catalog number: 25384-302 )
  5. Coplin jar
  6. Diamond marking pen
  7. Pasteur pipette
  8. Humid chamber
    Note: To make a humid chamber, place wet paper towels on the bottom of a large plastic dish with a lid.
  9. Metal rod (for macerating anthers)
  10. Microscopic glass slides (Thermo Fisher Scientific, catalog number: 12-550D )
  11. Acetic acid, 60% (Thermo Fisher Scientific, catalog number: A38-212 )
  12. BSA (bovine serum albumin, Sigma, catalog number: A9418 )
  13. Distilled H2O
  14. Vectashield (Vector Laboratories, catalog number: H-1200 )
  15. Ethanol
  16. Tween-20 (Sigma, catalog number: P9416 )
  17. Sodium citrate dihydrate (Sigma, catalog number: W302600 )
  18. Citric acid powder (Sigma, catalog number: W230618 )
  19. DAPI powder (4,6-diamidino-2-phenylindole, Vector Laboratories, catalog number: H-1200 )
  20. Onozuka R10 Cellulase (Grainger, catalog number: 31FX29 )
  21. Cytohelicase (Sigma, catalog number: 45-C8274 )
  22. Pectolyase from Aspergillus japonicus (Sigma, catalog number: P5936 )
  23. Sodium chloride (NaCl) (Sigma, catalog number: S7653 )
  24. Disodium phosphate (Na2HPO4) (Sigma, CAS number: 7558-79-4 )
  25. Monopotassium phosphate (KH2PO4) (Sigma, CAS number: 7778-77-0 )
  26. Potassium chloride (KCl) (Fisher Scientific, catalog number: p333-500 )
  27. Triton X-100 (Fisher Scientific, catalog number: 9002-93-1 )
  28. Blocking solution (see Recipes)
  29. Carnoy’s fixative (see Recipes)
  30. Citric buffer 10 mM pH 4.6 (see Recipes)
  31. Citric buffer 10 mM pH 6.0 (see Recipes)
  32. DAPI (see Recipes)
  33. Enzyme digestion mix (see Recipes)
  34. PBS (Phosphate-buffered saline) buffer (see Recipes)
  35. Phosphate-buffered saline + Triton X-100 (PBST) buffer (see Recipes)
  36. Tris-buffered saline buffer + Tween-20 (TBST) (see Recipes)

Equipment

  1. 37 °C incubator
  2. Forceps
  3. Dissecting microscope
  4. Fluorescence microscope
  5. Microwave oven
  6. Rocking platform shaker
  7. Warming plate (Premiere, model: XH-201 )

Procedure

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

Category

Plant Science > Plant cell biology > Cell imaging

Plant Science > Plant biochemistry > Protein

Cell Biology > Cell imaging > Fixed-tissue imaging

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