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Fluorescence Immunolocalization in Mitotic Chromosome Spreads from Maize Embryos

JL Jing-Han Liu*
YW Ya-zhong Wang*
LC Lin Chen*
YH Yan He

*Contributed equally to this work Published: Jun 20, 2019 DOI: 10.21769/BioProtoc.3279 Views: 3404

Edited by: Marisa Rosa

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Abstract

Immunolocalization is a necessary technique in the study of chromosome structure and function. It can provide important information for better understanding the relation between epigenetic modifications and chromosome condensation. In this protocol, we describe a way to localize and detect the distribution of histone H3 methylated at lysine 9 (inactive heterochromatin) in embryos at a period of 14 day-after-pollination (DAP). The protocol allows the observation of somatic cells in mitotic chromosome spreads.

Keywords: Immunolocalization search Mitotic search Maize search Plant search Embryo search Chromosome spread search

Background

Immunolocalization, a technique widely applied in cytogenetics, offers a great contribution to explore the structure of chromosomes. The development of cytogenetic techniques enables us to gain knowledge about spatial and temporal proceedings of a given protein in the whole cell (Fransz et al., 1998; De Paula and Techio, 2014). Epigenetic modifications regulate gene expression by affecting chromatin structure and function (Bannister and Kouzarides, 2011). Moreover, a number of studies indicated that pathways and patterns of epigenetic modification exhibit distinct properties in different species, including Arabidopsis, rice, and maize (Kim et al., 2009; Chen and Zhou, 2013; Capuano et al., 2014; Springer and Schmitz, 2017). Immunolocalization techniques benefit greatly to decipher the morphology and structure of chromosomes during each step of plant meiosis (Pawlowski et al., 2013). However, many proteins associated with chromatin are not expressed in tassels or ears during meiosis. Thus, somatic cells as materials were used to analyze the localization of proteins that are expressed in other plant tissues during mitosis. Analyzing the changes in those proteins and in epigenetic modifications between wild type and a mutant allows us to gain a deeper understanding in their functions. In summary, this method allows us to detect the spatial or temporal distribution of a given protein during mitosis, and identify the distribution of various epigenetic modifications attributing to the molecular function via mitotic chromosome spreads.

Materials and Reagents

  1. Pipette tips (Eppendorf)
  2. 2 ml Eppendorf tubes (Eppendorf)
  3. Scalpel
  4. 13 mm coverslips (CITOGLAS, catalog number: 10212020C)
  5. Glass slides (CITOGLAS, catalog number: 10127105P)
  6. Parafilm
  7. 35 mm Petri dish
  8. Staining jar
  9. Slide incubator box (Solarbio, YA0764)
  10. Plant Material: Maize plants from B73 pure inbred line 
  11. Paraformaldehyde concentration 16% in PBS (store at 4 °C) (Sigma, catalog number: P6148)
  12. Sodium chloride (NaCl) (Sigma, catalog number: S3014 )
  13. Autoclaved water
  14. KCl (Sigma, catalog number: P9541 )
  15. Na2HPO4 (Sigma, catalog number: S3264)
  16. KH2PO4 (Sigma, catalog number: P9791)
  17. NaOH (Sigma, catalog number: S5881)
  18. Poly-L-lysine solution (Sigma, catalog number: P8920)
  19. PIPES (Sigma, catalog number: V900404)
  20. EDTA (Sigma, catalog number: V900106)
  21. EGTA (Sigma, catalog number: E3889)
  22. Liquid nitrogen
  23. Spermine (Sigma, catalog number: S2876)
  24. Spermidine (Sigma, catalog number: S0266)
  25. DTT (Roche, catalog number: 3483-12-3)
  26. Tween®20 (Sigma, catalog number: P1379)
  27. BSA (VWR, catalog number: 0332)
  28. DAPI (Sigma, catalog number: D8417)
  29. Primary Antibodies
    DiMethyl-Histone H3-K9 Rabbit pAb antibody (Abclone, catalog number: A2359) (at a dilution of 1:50 in blocking buffer)
  30. Secondary Antibodies
    Alexa Fluor 555-conjugated Goat Anti-Rabbit IgG (H+L) antibody (Abclone, catalog number: AS058) (at a dilution of 1:100 in blocking buffer)
  31. 10x PBS (see Recipes)
  32. 10x Buffer A (see Recipes)
  33. Spermine stock (see Recipes)
  34. Spermidine stock (see Recipes)
  35. 2x Buffer A (see Recipes)
  36. Blocking Buffer (see Recipes)
  37. 1x Buffer A (see Recipes)

Equipment

  1. Pipette (Eppendorf)
  2. Vacuum pump
  3. Rotary shaker (Kylin-Bell Lab Instruments, ZD-2008)
  4. Tweezers
  5. Fluorescence Microscope (Nikon, model: Eclipse Ci-S)
  6. Super High Pressure Mercury Lamp (Nikon, model: C-SHG1)
  7. Incubator (Yamato, IC412C)

Software

  1. ImageJ64 software (National Institute of Health)
  2. Nis-Elements F64-bit (Nikon)

Procedure

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

Category

Plant Science > Plant molecular biology > DNA > DNA modification

Plant Science > Plant molecular biology > Protein

Molecular Biology > DNA > Chromosome engineering

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