Materials and Reagents

0.2 μm sterile syringe filter (Corning, catalog number: 431219 )
Microscope slide and coverslip
Needle (30 gauge)
3 mm Whatman filter paper (GE Healthcare, Dharmacon, catalog number: 1006090 )
Note: Currently, it is “Sigma-Aldrich, catalog number: 1006090”.
Mineral oil (Sigma-Aldrich, catalog number: M5904 )
Paraformaldehyde (Sigma-Aldrich, catalog number: F8775 )
25% glutaraldehyde (Junsei, catalog number: 273721250 )
Sodium carcodylate (Ted Pella, catalog number: 18851 )
Potassium phosphate monobasic (KH₂PO₄) (Sigma-Aldrich, catalog number: P5379 )
Ethanol (Merck Millipore Corporation, Calbiochem^®, catalog number: 1.00983.1011 )
Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: 30620 )
Osmium tetroxide (OsO₄) (Ted Pella, catalog number: 96049 )
Spurr resin (Ted Pella, catalog number: 183004221 )
Double end mold (Ted Pella, catalog number: 10590 )
Toluidine blue O (Sigma-Aldrich, catalog number: T3260 )
EDTA (BioShop, catalog number: EDT001.500 )
Triton X-100 (Sigma-Aldrich, catalog number: T8532 )
Note: This product has been discontinued.
4’, 6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich, catalog number: 32670 )
Acetic acid (Merck Millipore Corporation, Calbiochem^®, catalog number: 1.00063.1011 )
1/2 strength Karnovsky’s fixative solution (see Recipes)
Toluidine blue O solution (see Recipes)
Fixative solution (see Recipes)
DAPI solution for developing pollen (see Recipes)
DAPI solution for mature pollen grain (see Recipes)

Equipment

Ultramicrotome (Boeckeler Instruments, model: MT990 motorized precision microtome )
Hot plate
Water bath
Incubator
Fume hood
Light and fluorescence microscope (Leica Microsystems, model: DM2500 )
Vacuum pump and vacuum jar
Glass pipette

Procedure

Semi-thin sectioning of Arabidopsis floral buds for light microscopy

Embedding of Arabidopsis floral buds

Collect the whole inflorescence in a 2 ml tube.
Add 1 ml of 1/2 strength Karnovsky’s fixative solution (Karnovsky, 1965) into the tube and cover it with 3MM Whatman filter paper beneath the surface of the solution.
Incubate the tube in a vacuum jar under 400 mmHg for 30 min.
Incubate the tube at 4 °C for 4 h to overnight.
Wash the samples three times with 1 ml of 0.1 M sodium carcodylate (pH 7.4) for every 5 min on the rocker with gentle agitation.
Carry out a post-fixation treatment for overnight at 4 °C with 1 ml of 1% osmium tetroxide using a glass pipette.
Note: If you want to do immuno-staining, do not perform post-fixation with osmium tetroxide. This can inhibit the binding of antibody to antigen.
Dehydrate the sample with 10, 20, 30, 40, 50, 60, 70, 80, and 90% ethanol for 30 min for each treatment sequentially and with 100% ethanol for overnight.
Prepare medium spurr resin according to manufacturer’s protocol in the hood and then incubate the spurr resin in a vacuum jar for 30 min to eliminate air bubbles.

Infiltrate the spurr resin prepared at step A1-h to the dehydrated sample at step A1-g and remove the residual ethanol of the sample by the following procedures.

Step	100 % Ethanol : Pure resin	Time (h)
①	3 : 1	4
②	1 : 1	4
③	1 : 3	4
④	− : 4	Overnight

Fill the bottom of the double end mold tray with spurr resin, and then incubate it at 58 °C for overnight to make drop panel.
Immerse the sample in the mold tray with spurr resin, and then incubate it at 58 °C for 3 days.
Take out the block from the mold tray and keep the sample at room temperature.

Semi-thin sectioning of the sample embedded in the spurr resin.
1. Trim the resin block ends with a razor blade to make the shape of trapezoid centering around a specimen (Figure 1).
  
  Figure 1. Resin blocks embedded specimens. A. Arabidopsis inflorescences embedded in solidified resin. B. Trimmed resin by razor blade.
2. Fix the resin block to the head of the microtome.
3. Cut 1-10 μm transverse sections of the resin-embedded specimens using an MT-990 motorized precision microtome with the glass knives. Four μm transverse sections were made in the case of Borg et al. (2009).
4. Collect the floating samples in water on to a glass slide with a platinum wire and put the glass slide with the samples on a hot plate at 50 °C for 5 min.
5. Drop 100 μl of the toluidine blue solution on the surface of the sections by using a syringe, and then dry it on the hot plate at 50 °C for 5 min.
6. Wash the samples with sprinkled water using a syringe, and then dry the excess moisture on the slide put on the hot plate at 50 °C.
7. Drop 20 μl of the mineral oil and cover it with a coverslip, then seal the edges of a coverslip with nail varnish (Figure 2).
  
  Figure 2. Sections stained with toluidine blue solution on a slide with a coverslip

Fluorescence microscopic analysis of developing microspore and mature pollen with 4', 6-diamidino-2-phenylindole (DAPI)
1. Preparation of mature pollen grains with DAPI staining.
  1. Fix the whole inflorescence with opened flowers in a fixative solution (Oh et al., 2010) for 5 min.
    Note: If you want to detect fluorescent proteins or GUS staining, skip this procedure.
  2. Collect 3-4 opened flowers at flower stage 13-14 from Arabidopsis (Sanders et al., 1999) in a 1.5 ml tube containing 200 μl of DAPI solution (Park et al., 1998).
  3. Vortex the tube briefly at maximum speed for 10 sec to separate pollen grains from the dehiscent anther wall.
  4. Incubate the tube in the dark at room temperature for 30 min.
  5. Transfer the supernatant to a new 1.5 ml tube with a 200 μl pipette man.
    Note: Do not centrifuge. Do not transfer any other floral organs except pollen grains in the supernatant.
  6. Concentrate the pollen grains by centrifuging at 13,000 rpm for 30 sec at room temperature, and then discard the supernatant.
  7. Resuspend the pollen pellets in 5-10 μl of DAPI solution.
  8. Mount this sample on the microscope slide under the coverslip.
2. Preparation of developing microspores from anthers with DAPI staining
  1. Fix the whole inflorescence with opened flowers in a fixative solution (Oh et al., 2010) for 5 min.
    Note: If you want to detect fluorescent proteins or GUS staining, skip this procedure.
  2. Dissect the single anther from inflorescence on a microscope slide with a needle while adding 4-5 μl of 0.3 M mannitol or DAPI solution (Park et al., 1998).
  3. Collect free microspores on a slide in a 1.5 ml tube and incubate them in the dark for 30 min.
  4. Mount this sample on the microscope slide under the coverslip.
3. Acquire images with a camera affixed to a Leica DM2500 microscope under the epifluorescence channel (15% of 405 nm/430-470 nm for DAPI, 470 nm/530 nm for GFP and 561 nm/570-610 nm for DsRed) (Figure 3) (Kim et al., 2015).
  
  Figure 3. Image of transverse sections of Arabidopsis anther stained with toluidine blue solution by light microscopy according to the protocol

Recipes

1/2 strength Karnovsky’s fixative solution
2% paraformaldehyde
2.5% glutaraldehyde
0.1 M sodium cacodylate (pH 7.4)
Prepare this solution freshly prior to use
Toluidin blue O solution
Dissolve powder in 1 or 2 ml of water to make final 0.05% solution, and filter this solution through a 0.2 μm sterile syringe filter. Store this solution at 4 °C protected from light.
Fixative solution
Ethanol: acetic acid=3:1
Prepare this solution freshly prior to use.
DAPI solution for developing pollen
0.1 M sodium phosphate (pH 7.0)
1 mM EDTA (pH 8.0)
0.1% Triton X-100
1 μg/ml DAPI
Store this solution at 4 °C protected from light
DAPI solution for mature pollen grain
0.1 M sodium phosphate (pH 7.0)
1 mM EDTA, pH 8.0
0.1% Triton X-100
0.4 μg/ml DAPI
Store this solution at 4 °C protected from light

Acknowledgments

This study was supported by grants from the Next-Generation BioGreen 21 Program (PJ01104701), Rural Development Administration, Republic of Korea and Basic Science Research Programs through the National Research Foundation of Korea funded by the Ministry of Education (no. 2013R1A1A2062335). This protocol was based on Kim et al. (2015).

References

Berger, F. and Twell, D. (2011). Germline specification and function in plants. Annu Rev Plant Biol 62: 461-484.
Borg, M., Brownfield, L. and Twell, D. (2009). Male gametophyte development: a molecular perspective. J Exp Bot 60(5): 1465-1478.
Karnovsky, M. J. (1965). A formaldehyde-glutaraldehyde fixative of high osmolarity for use in electron microscopy. J Cell Biol 27(2): 137A-138A.
Kim, M. J., Kim, M., Lee, M. R., Park, S. K. and Kim, J. (2015). LATERAL ORGAN BOUNDARIES DOMAIN (LBD)10 interacts with SIDECAR POLLEN/LBD27 to control pollen development in Arabidopsis. Plant J 81(5): 794-809.
McCormick, S. (2004). Control of male gametophyte development. Plant Cell 16 Suppl: S142-153.
Oh, S. A., Park, K. S., Twell, D. and Park, S. K. (2010). The SIDECAR POLLEN gene encodes a microspore-specific LOB/AS2 domain protein required for the correct timing and orientation of asymmetric cell division. Plant J 64(5): 839-850.
Park, S. K., Howden, R. and Twell, D. (1998). The Arabidopsis thaliana gametophytic mutation gemini pollen1 disrupts microspore polarity, division asymmetry and pollen cell fate. Development 125(19): 3789-3799.
Sanders, P. M., Bui, A. Q., Weterings, K., McIntire, K. N., Hsu, Y. C., Lee, P. Y., Truong, M. T., Beals, T. P. and Goldberg, R. B. (1999). Another developmental defects in Arabidopsis thaliana male-sterile mutants. Sex Plant Reprod 11(6): 297-322.