Microscopic Observation of Subcellular GFP-tagged Protein Localization in Rice Anthers

Materials and Reagents

1. Micropipette tips
2. Razor blade (FEATHER Safety Razor, catalog number: 99027 )
3. 2 ml Safe-Lock microtube (Eppendorf, catalog number: 0030120094 )
4. Microscope slide glass (Matsunami Glass, catalog number: SMAS-01 )
5. Coverslip (Matsunami Glass, catalog number: C218181 )
6. CRYO DISH No2 (SHOEI WORK’S, catalog number: 1101-2 )
7. (Optional) 15 ml centrifuge tubes (TPP Techno Plastic Products, catalog number: 91015 )
8. (Optional) Nylon filter mesh sheet
9. Young panicles at a preferred developmental stage from transgenic rice plants expressing fluorescent proteins (if you target meiotic stages in japonica rice varieties, use 4-8 cm long panicles containing flowers with 0.5-0.9 mm long anthers)
10. Low melting temperature agarose (SeaPlaque^TM Agarose) (Lonza, catalog number: 50101 )
11. ddH₂O
12. Krazy Glue^® (Krazy Glue, catalog number: KG585 )
13. VECTASHIELD mounting medium with DAPI (Vector Laboratories, catalog number: H-1200 , store at 4 °C in the dark condition)
14. Nail polish
15. (Optional) Paraformaldehyde (Wako Pure Chemical Industries, catalog number: 162-16065 )
16. (Optional) 5 N NaOH solution
17. (Optional) Ice
18. (Optional) PIPES (Wako Pure Chemical Industries, catalog number: 345-02225 )
19. (Optional) EGTA (Wako Pure Chemical Industries, catalog number: 342-01314 )
20. (Optional) MgSO₄•7H₂O (Wako Pure Chemical Industries, catalog number: 138-00415 )
21. (Optional) KOH solid (60 g or more, Wako Pure Chemical Industries, catalog number: 168-21815 )
22. (Optional) Glycerol (Wako Pure Chemical Industries, catalog number: 075-00616 )
23. (Optional) DMSO (Wako Pure Chemical Industries, catalog number: 048-21985 )
24. (Optional) 5x PMEG stock buffer (see Recipes, store at 4 °C. It is available for 6 months)
    

Equipment

1. Micropipettes
2. (Optional) Measuring cylinder
3. 100 ml polypropylene beaker
4. (Optional) 200 ml or 300 ml flask
5. Standard microwave oven
6. Block incubator (ASTEC, model: BI-525A )
7. Stereo microscope (Nikon, model: SMZ445 )
8. Fine forceps (Fine Science Tools, DUMONT, model: #5 )
9. Vibrating microtome (MicroSlicer) (DOSAKA EM, model: ZERO-1 )
10. Confocal Laser Scanning Microscope (CLSM, Olympus, model: FV300 ) or fluorescent microscope
11. (Optional) Autoclave
12. (Optional) Vacuum Pressure pump (e.g., Merck, model: WP6122050 )
13. (Optional) Vacuum desiccator (e.g., SP Scienceware - Bel-Art Products - H-B Instrument, catalog number: F42400-2041 )
14. (Optional) Sample Shaker
15. (Optional) Water bath
    

Software

1. ImageJ (or Fiji)
2. Photoshop CC (Adobe)
   

Procedure