Cytology and Microscopy: Immunolocalization of Covalently Modified Histone Marks on Barley Mitotic Chromosomes

Materials and Reagents

1. Sterilin^TM 9 cm petri dish
(Thermo Fisher Scientific, catalog number: 1011RR )
   
2. Whatman^® 9 cm filter paper (Sigma-Aldrich, catalog number: Z240079 )
   
3. Fine marker pen permanent (STABILO International GmbH)
   
4. HB graphite pencil
   
5. 15 ml centrifuge tube (any brand)
   
6. Single edge razor blade (Thermo Fisher Scientific, catalog number: 11904325 )

   
7. 1.5 ml microcentrifuge tubes
(VWR International, catalog number: 89000-028 )
   
8. Microscope Polysine^® slide (Thermo Fisher Scientific, Menzel Glaser, catalog number: 10143265 ) or Superfrost^® plus micro slide (VWR International, catalog number: 48311-703 )
   Note: Currently, it is “Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 10143265 ”.
   
9. Borosilicate glass coverslip, 25 x 50, thickness 0.13 mm (VWR International, catalog number: 631-0137 )
   
10. Tissue paper
    
11. PAP pen for immunostaining (Sigma-Aldrich, catalog number: Z377821 )
    
12. 0.45 μm, 26 mm diameter syringe filter (Cole-Parmer, catalog number: UY-02915-58 )
    
13. Glass Coplin staining jar (any brand)
    
14. Seeds from barley cv. Morex
    
15. Immersion oil, Immersol^TM W 2010 (Carl Zeiss)
    
16. Autoclaved distilled water (Barnstead^® E-pure 4 modules cartridges)
    
17. 5% bleach (Domestos)
    
18. 70% ethanol

    
19. Liquid nitrogen
    
20. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A7030 )
    
21. Antibodies
    1. Primary antibodies: anti-trimethyl-histone H3 (Lys27) antibody (H3K27me3) (Merck Millipore Corporation, catalog number: 07-449 ), anti-monomethyl-histone H3 (Lys27) antibody (K3K27me1) (Merck Millipore Corporation, catalog number: 07-448 ) and histone H3K9me2 antibody (pAb) (H3K9me2) (Active Motif, catalog number: 39375 )
       Note: Currently, it is “EMD Millipore Corporation, catalog numbers: 07-449 and 07-448 ”.
    2. Secondary antibodies: goat anti-Rabbit IgG (H+L), Alexa Fluor^® 488 conjugate (Thermo Fisher Scientific, catalog number: A-11034 )
22. Hoechst 33342 (10 mg/ml) (Thermo Fisher Scientific, Molecular Probes^TM, catalog number: H3570 )

    
23. Vectashield antifade mounting medium (Vector Laboratories, catalog number: H-1000 )
    
24. Clear nail varnish (Technic)
    
25. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 )
    
26. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9541 )
    
27. Sodium phosphate dibasic (Na₂HPO₄) (Sigma-Aldrich, catalog number: 255793 )
    
28. Potassium phosphate monobasic (KH₂PO₄) (Sigma-Aldrich, catalog number: P5379 )
    
29. Tween^® 20 (Sigma-Aldrich, catalog number: P9416 )
    
30. Triton^TM X-100 (Sigma-Aldrich, catalog number: T8787 )
    
31. Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: S8045 )
    
32. Paraformaldehyde (Sigma-Aldrich, catalog number: P6148 ) 

    
33. Cellulase Onozuka R10 (Duchefa Biochemie, catalog number: C8001 ) 

    
34. Pectolyase Y23 (Dushefa Biochemie, catalog number: P8004 ) 

    
35. 10x PBS (see Recipes)
    
36. 1x PBS, 0.5% Triton^® X-100 (see Recipes)
    
37. 1 M NaOH (see Recipes)
    
38. 4% formaldehyde (see Recipes)
    
39. 0.1 M citric acid (see Recipes)
    
40. 0.1 M sodium citrate (see Recipes)
    
41. Citrate buffer (see Recipes)
    
42. Enzyme mixture (see Recipes)
    
43. Blocking solution (see Recipes)

Equipment

1. Optoelectronically controlled small orbital shaker, 2,200 rpm, 230 V (IKA, model: VXR basic Vibrax^® )
   
2. Stereomicroscope (optional) (TEC Microscopes LTD., model: HM-4 )
   
3. Light microscope (TEC Microscopes LTD., model: LM-2TR )
   
4. Confocal microscope LSM 710 (Carl Zeiss)
   
5. Objective (for Zeiss LSM 710): 60x APO C-apochromat 63x/1.20 W Korr M27
   
6. DAPI fluorescence (blue 405, UV lamp) (Hoechst)
   
7. Fitted 405 nm diode laser (for Hoechst acquisition on the LSM 710)
   
8. Fitted 488 argon laser (for Alexa Fluor^® 488 acquisition on the LSM 710)
   
9. Moisture or wet chamber
   Note: Commercially available for immunohistochemistry application or home made by using a plastic box lined with wet paper and wax sheet or glass rod to keep the slides out of direct contact with water (Figure 1). The chamber needs to be closed so the humidity is preserved during the protocol.
   
10. Aluminium slide tray, 20 slides capacity (Brunel Microscopes Ltd.)
    
    
    Figure 1. Wet chamber. The principle of the wet chamber is to keep the sample moist at all times.

Software

1. Zen 2010 software (Carl Zeiss, version 6.0)

   
2. Fiji (Image J, version 1.49m) (Schindelin et al., 2012) with deconvolution package (EPFL, Biomedical Imaging Group, Switzerland)
   
3. Imaris (optional) (Bitplane AG, version 8.1.2)
   

Procedure