Materials and Reagents

Immunogold labeling of microtubules and microtubule organizing center proteins
1. Scintillation vials with aluminum covered caps (Fisher Scientific, catalog number: 03-340-4B)
  Manufacturer: DWK Life Sciences, Kimble, catalog number: 7450320 .
2. Pasteur pipette (Fisher Scientific, catalog number: 13-678-20C )
3. Gelatin capsules (Electron Microscopy Sciences, catalog number: 70100 )
4. 200 mesh nickel grids (Electron Microscopy Sciences, catalog number: EMS200-Ni )
5. 300 mesh gold grids (Electron Microscopy Sciences, catalog number: EMS300-Au )
6. Glass Petri dishes (Corning, catalog number: 3160-101 )
7. 90 mm filter paper (GE Healthcare, catalog number: 1004-090 )
8. Sterile filters (0.2 µm) (Corning, catalog number: 431212 )
9. Parafilm (Sigma-Aldrich, Parafilm, catalog number: P7793 )
10. Dental wax plate (Electron Microscopy Sciences, catalog number: 72670 )
11. Glass slides (Fisher Scientific, catalog number: 12-544-1 )
12. Microcentrifuge tubes, 1.5 ml natural (USA Scientific, catalog number: 1615-5500 )
13. Ethylene dichloride (Electron Microscopy Sciences, catalog number: 13250 )
14. LR white resin (Electron Microscopy Sciences, catalog number: 14383 )
15. Primary antibodies: (see Table 1), centrin (Sigma-Aldrich, catalog number: ABE480 ); MPM-2 (EMD Millipore, catalog number: 05-368 )
  
  Table 1. Primary antibodies used to immunogold label microtubules, microtubule organizing center proteins, and carbohydrates and arabinogalactan proteins in extracellular matrices during spermatogenesis
  
  ^aPhaeoceros carolinianus (hornwort); ^bPhyscomitrella patens (moss); ^cAulacomnium palustre (moss); ^dCeratopteris richardii (fern), and ^eGinkgo biloba (seed plant).
16. Secondary antibody: goat anti-mouse conjugated with gold (Millipore Sigma, catalog number: G7652 )
17. Sorenson’s phosphate buffer, 0.2 M, pH 7.2 (Electron Microscopy Sciences , catalog number: 11600-10 )
18. Glutaraldehyde (Electron Microscopy Sciences, catalog number: 16120 )
19. Osmium tetroxide (Electron Microscopy Sciences, catalog number: 19150 )
20. PIPES buffer, 0.2 M, pH 7.2 (Sigma-Aldrich, catalog number: P6757 )
21. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: B4287 )
22. Uranyl acetate (Polyscience, catalog number: 21447 )
23. Lead nitrate (Electron Microscopy Sciences, catalog number: 17900 )
24. Sodium citrate (Electron Microscopy Sciences, catalog number: 21140 )
25. 1 N NaOH (Electron Microscopy Sciences, catalog number: 21170-01 )
26. 0.01 M phosphate buffer (pH 7.2) (see Recipes)
27. 0.05 M phosphate buffer (pH 7.2) (see Recipes)
28. 2.5% glutaraldehyde (see Recipes)
29. 2% aqueous osmium tetroxide (see Recipes)
30. 0.02 M phosphate buffer (pH 7.2) (see Recipes)
31. Antibodies (see Recipes)
32. 2% PBS/BSA (see Recipes)
33. 0.05 M PIPES buffer (pH 7.2) (see Recipes)
34. 2% uranyl acetate (see Recipes)
35. 1 N NaOH (see Recipes)
36. Reynold’s lead citrate (see Recipes)
Immunogold labeling of spermatozoid matrix and cell wall constituents
1. Scintillation vials with aluminum covered caps (Fisher Scientific, catalog number: 03-340-4B)
  Manufacturer: DWK Life Sciences, Kimble, catalog number: 7450320 .
2. Pasteur pipette (Fisher Scientific, catalog number: 13-678-20C )
3. 200 mesh nickel grids (Electron Microscopy Sciences, catalog number: EMS200-Ni )
4. 300 mesh gold grids (Electron Microscopy Sciences, catalog number: EMS300-Au )
5. 90 mm filter paper (GE Healthcare, catalog number: 1004-090 )
6. Dental wax plate (Electron Microscopy Sciences, catalog number: 72670 )
7. Glass Petri dish (Corning, catalog number: 3160-101 )
8. Glass Slides (Fisher Scientific, catalog number: 12-544-1 )
9. Gelatin capsules (Electron Microscopy Sciences, catalog number: 70100 )
10. Microcentrifuge tubes, 1.5 ml natural (USA Scientific, catalog number: 1615-5500 )
11. Parafilm (Sigma-Aldrich, Parafilm, catalog number: P7793 )
12. Sterile filters (0.2 µm) (Corning, catalog number: 431212 )
13. Ethylene dichloride (Electron Microscopy Sciences, catalog number: 13250 )
14. LR white resin (Electron Microscopy Sciences, catalog number: 14383 )
15. Primary antibodies (PlantProbes) (see Table 1)
16. Secondary antibody: Goat-Anti-Rat IgG-gold (Sigma-Aldrich, catalog number: G7035 )
17. Secondary antibody: goat anti-mouse conjugated with gold (Sigma-Aldrich, catalog number: G7652 )
18. Sorensen’s phosphate buffer, 0.2 M, pH 7.2 (Electron Microscopy Sciences , catalog number: 11600-10 )
19. Glutaraldehyde (Electron Microscopy Sciences, catalog number: 16120 )
20. Osmium tetroxide (Electron Microscopy Sciences, catalog number: 19150 )
21. PIPES buffer, 0.2 M, pH 7.2 (Sigma-Aldrich, catalog number: P6757 )
22. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: B4287 )
23. Uranyl acetate (Polyscience, Inc., catalog number: 21447 )
24. Lead nitrate (Electron Microscopy Sciences, catalog number: 17900 )
25. Sodium citrate (Electron Microscopy Sciences, catalog number: 21140 )
26. 1 N NaOH (Electron Microscopy Sciences, catalog number: 21170-01 )
27. 0.01 M phosphate buffer (pH 7.2) (see Recipes)
28. 0.05 M phosphate buffer (pH 7.2) (see Recipes)
29. 2.5% glutaraldehyde (see Recipes)
30. 2% aqueous osmium tetroxide (see Recipes)
31. 0.02 M phosphate buffer (pH 7.2) (see Recipes)
32. Antibodies (see Recipes)
33. 2% PBS/BSA (see Recipes)
34. 0.05 M PIPES buffer (pH 7.2) (see Recipes) (see Recipes)
35. 2% uranyl acetate (see Recipes)
36. 1 N NaOH (see Recipes)
37. Reynold’s lead citrate (see Recipes)

Equipment

Immunogold labeling of microtubules and microtubule organizing center proteins
1. Bunsen burner (Fisher Scientific, catalog number: S12809 )
2. -20 °C freezer
3. M2100 Benchmark Tube Rocker (Benchmark Scientific Inc.)
4. Oven (General Signal, model: Gravity convection )
5. Water bath (Sheldon Manufacturing, model: SWB23 )
6. Micropipette (Bioexpress, GeneMate, catalog number: P-4963-20 )
7. Centrifuge (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 75004061 )
8. Diamond knife (Diatome, specs: Ultra, 45°, 4 mm, Wet)
9. Transmission electron microscope (Hitachi, model: HF7100 )
Immunogold labeling of spermatozoid matrix and cell wall constituents
1. Bunsen burner (Fisher Scientific, catalog number: S12809 )
2. M2100 Benchmark Tube Rocker (Benchmark Scientific Inc.)
3. Oven (General Signal, model: Gravity convection )
4. Micropipette (Bioexpress, catalog number: P-4963-20 )
5. Centrifuge (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 75004061 )
6. Diamond knife (Diatome, specs: Ultra, 45°, 4 mm, Wet)
7. Humid chamber (see Notes)
8. Transmission electron microscope (Hitachi, model: HF7100 )

Procedure

Immunogold labeling of microtubules and microtubule organizing center proteins (Vaughn and Bowling, 2008; Vaughn, 2013)
Note: For each solution change described below, completely saturate samples by adding solution until it covers over the material in the vial/tube.
1. Fix small pieces of several antheridia or whole gametophyte plants (if small, like those of ferns) in 2.5% glutaraldehyde in 0.05 M Sorenson’s phosphate buffer (pH 7.2) at room temperature. Scintillation vials with aluminum-covered caps are ideal for this process (alternatively, you can also use 1.5 ml microcentrifuge tubes). If parts of plants are used, do all cutting of samples in a drop of the glutaraldehyde solution.
2. Wash in the same 0.05 M Sorenson’s buffer, 2-3 exchanges, 15 min each at 4 °C. To facilitate exchanging solutions without losing the small samples, the tip of the Pasteur pipette is heated over a Bunsen burner so that the tip aperture is down to about 1/3 of its original size. In this way, clean exchanges may be made so that the tiny samples are not sucked up into the pipette and lost during the exchanges.
3. Post-fix tissue for 10 min in 2% (v/v) osmium.
4. Serial dehydration: Remove the PIPES buffer and add 25% ethanol (in 0.05 M PIPES buffer) to the vial, increasing the percentage of ethanol by 25% increments until 75% ethanol is reached.
5. Exchange the solution with 100% ethanol several times. These steps should be at 4 °C, and the vials transferred to a -20 °C freezer after the second 100% ethanol transfer.
6. Add LR White resin (London Resin Company) to reach 25% plastic (in ethanol). Shake vial to mix plastic into the solvent and maintain at -20 °C for one day. Repeat this procedure by adding plastic to 50% for one day and then add plastic to 75% for another day. Exchange the solutions, putting 100% resin in the vial. Shake the vial and make sure the samples have sunk in the resin. Allow them to spend 24 h in 100% resin.
7. Allow the vials to warm to room temperature and shake on a rocking platform for 24 h. This will allow for infiltration of the resin throughout the sample.
8. Transfer the samples to gelatin capsules and fill the capsule with 100% resin forming a meniscus. Rotate the cap as it is inserted to create an oxygen-excluding shield. Place in a 50 °C oven and remove in ~2 d.
9. Trim the samples and cut 100 nm sections (pale gold reflectance colors) with a diamond knife and fume the sections with ethylene dichloride to stretch the sections. Pick up the sections by touching the dull side of 300 mesh gold grids or 200 mesh nickel grids.
10. Place mounted grids in a closed CLEAN Petri dish and dry for 1-2 h at room temperature or place the Petri dish on a slide warmer (45-50 °C) for 30 min.
11. Float the grids, section-side down on 10 µl drops of the following solutions (all of which are filtered through 250 nm filters) and times:
  1. 2% BSA in 0.02 M sodium phosphate buffer (PBS/BSA; pH 7.2), 30 min (BLOCKING).
  2. On drops of primary antibodies or monoclonal antibodies diluted from 1:20 in 2% PBS/BSA for 3-4 h at room temperature.
12. Rinse grids by placing them on drops of 2% BSA/PBS 4 x 3 min.
13. Place grids on 5-15 µl drops of secondary gold-labeled antibody or Protein A/gold diluted 1/20-in 2% BSA/PBS in ‘humid chamber’ 1-3 h.
14. Move grids to 5-10 µl drops of 0.02 M PBS for rinsing 4 x 3-5 min (FILTER and CENTRIFUGE).
15. Rinse each grid with a jet of double distilled autoclaved water that has been filtered (0.2 µm) and let it dry.
16. Post-stain the grids for 2 min in 2% uranyl acetate, wash in double distilled autoclaved water and post-stain in Reynold’s lead citrate. Wash in double distilled autoclaved water, dry and examine under transmission electron microscope.
Immunogold labeling of spermatozoid matrix and cell wall constituents (Lopez and Renzaglia, 2014)
1. Fix small pieces of antheridial tissue or whole gametophyte plants (if small, like those of ferns) in 2.5% glutaraldehyde in 0.05 M Sorenson’s phosphate buffer (pH 7.2) at room temperature. Scintillation vials with aluminum-covered caps are ideal for this process (alternatively, you can also use 1.5 ml microcentrifuge tubes). If parts of plants are used, do all cutting of samples in a drop of the glutaraldehyde solution.
2. Wash in the same 0.05 M Sorenson’s buffer, 2-3 exchanges, 15 min each at 4 °C. To facilitate exchanging solutions without losing the small samples, the tip of the Pasteur pipette is heated over a Bunsen burner so that the tip aperture is down to about 1/3 of its original size. In this way, clean exchanges may be made so that the tiny samples are not sucked up into the pipette during the exchanges.
3. Post-fix tissue for 10 min in 1% (v/v) osmium tetroxide (OsO₄).
4. Rinse tissue 3 x in distilled water (10 min each).
5. Serial dehydration: Remove the water and add 25% ethanol (in water) to the vial, increasing the percentage of ethanol by 25% increments until 75% ethanol is reached.
6. Exchange the solution 3 x with 100% ethanol.
7. Add LR White resin (London Resin Company) to reach 25% plastic (in ethanol). Shake vial to mix plastic into the solvent and maintain at -20 °C for one day. Repeat this procedure by adding plastic to 50% for one day and then add plastic to 75% for another day. Exchange the solutions, putting 100% resin in the vial. Shake the vial and make sure the samples have sunk in the resin. Allow them to spend 24 h in 100% resin.
8. Allow the vials to warm to room temperature and shake on a rocking platform for 24 h. This will allow for infiltration of the resin throughout the sample.
9. Transfer the samples to gelatin capsules and fill the capsule with 100% resin forming a meniscus. Rotate the cap as it is inserted to create an oxygen-excluding shield. Place in a 50 °C oven and remove in ~2 d.
10. Trim the samples and cut 90-100 nm sections (pale gold reflectance colors) with a diamond knife and fume the sections with ethylene dichloride to stretch the sections. Pick up the sections by touching the dull side of 300 mesh gold grids or 200 mesh nickel grids.
11. Place mounted grids on a clean piece of filter paper in a closed CLEAN Petri dish and dry for 1-2 h at room temperature or place the Petri dish on a slide warmer (45-50 °C) for 30 min.
12. Using a humid chamber (see Note 2) place a piece of dental wax on completely wet filter paper, float the grids, section-side down on 10 µl drops in the following solutions (all of which are centrifuged and filtered through 250 nm filters) and times:
  1. 2% BSA in 0.05 M sodium phosphate buffer (BSA/PBS; pH 7.2), overnight (BLOCKING).
  2. On drops of primary antibodies diluted from 1:20 in 2% BSA/PBS for 3-4 h at room temperature. Alternatively, grids can be left in primary antibody overnight at 4 °C.
13. Rinse grids by placing them on drops of 2% BSA/PBS 4 x 3 min.
14. Place grids on 10 µl drops of secondary gold-labeled antibody diluted 1/20 in 2% BSA/PBS in ‘humid chamber’ 1-3 h.
15. Move grids to 10 µl drops of 0.02 M PBS (FILTER and CENTRIFUGE) for rinsing 4 x 3-5 min.
16. Rinse each grid with a jet of double distilled autoclaved water that has been filtered (0.2 µm) and let it dry.
17. Post-stain the grids for 2 min in 2% uranyl acetate, wash in double distilled autoclaved water and post-stain in Reynold’s lead citrate for 30 sec. Wash in double distilled autoclaved water, dry and examine under transmission electron microscope.

Notes

Osmium tetroxide (OsO₄) is a heavy fixative that stains lipids in membranous structures and vesicles. Additionally, the reduced heavy metal adds density and contrast to biological tissue.
Humid chamber is a Petri dish, whose bottom is covered with a fresh, clean wet filter paper and cover is sealed with Parafilm if sitting overnight. Aliquots of BSA/PBS can be kept frozen until used. Centrifuge prior to use and store refrigerated (4 °C). 1-2% BSA/PBS can be divided in small aliquots (100-200 µl) for each time of labeling and kept at -20 °C, up to 6 months.
The primary antibody in BSA/PBS should not be frozen and thawed repeatedly; it decreases the strength of enzyme.
Use completely clean tools and Petri dishes during the labeling process.
Millipore filter or centrifuge every single agent and only draw from supernatant, use sterile filtered (0.2 µm) water for rinses after PBS, lead citrate and uranyl acetate stains.
Aliquots (40-50 µl) of primary antibody must be kept at -20 °C.
Secondary antibody with gold particles must be kept at 4 °C, never place in freezer.
After labeling agents are thawed (aliquots) do not freeze them again. They are good for maximum 3-4 days at 4 °C.
Drop size for BSA/PBS as well as other liquids could be between 5 to 10 µl, if you are stopping for a step overnight use 10 µl.
If you stop at any of the above steps (prior to putting grids in the secondary antibody) it should not be more than overnight and humid chamber should be sealed and kept in the refrigerator.
Note: Grids should not be left in the secondary antibody overnight because it will increase the amount of background ‘noise’ (i.e., over labels).
It is recommended to wick grids on a small piece of filter paper prior to moving them to a new solution and between different steps of the same rinses.
Use a new piece of Parafilm (or rinse and wipe dental wax) each time you move grids to a new solution.
Glass Petri dishes are preferred. Plastic creates static electricity and causes the grids to cling to the top of the dish.

Recipes

0.01 M phosphate buffer (pH 7.2)
Add 5 parts 0.20 M Sorenson’s phosphate buffer (pH 7.2) to 95 parts double distilled autoclaved water
0.05 M phosphate buffer (pH 7.2)
Add 25 parts 0.20 M Sorenson’s phosphate buffer (pH 7.2) to 75 parts double distilled autoclaved water
2.5% glutaraldehyde
1. Add 1 part 10% glutaraldehyde to 1 part double distilled autoclaved water
2. Add 1 part 0.20 M Sorensen’s phosphate buffer (pH 7.2) to 1 part double distilled autoclaved water
3. Add 1 part 5% glutaraldehyde to 1 part 0.10 M Sorensen’s phosphate buffer (pH 7.2)
2% aqueous osmium tetroxide
Add 1 part 4% aqueous osmium tetroxide to 1 part double distilled autoclaved water
0.02 M phosphate buffer (pH 7.2)
Add 1 part 0.20 M Sorenson’s phosphate buffer (pH 7.2) to 10 parts double distilled autoclaved water
Antibodies
Add 1 part antibody to 20 parts 2% PBS/BSA
2% PBS/BSA
Add 0.20 g of bovine serum albumin to 10 ml 0.02 M phosphate buffer (pH 7.2)
0.05 M PIPES buffer (pH 7.2)
Add 1 part 0.30 M PIPES buffer (pH 7.2) to 6 parts double distilled autoclaved water
2% uranyl acetate
Add 1 g uranyl acetate in 50 ml double distilled autoclaved water
1 N NaOH
Dissolve 40.00 g sodium hydroxide in water to make volume 1 L
Reynold’s lead citrate
1. Add 1.33 g lead nitrate and 1.76 g sodium citrate to 30 ml boiled double distilled autoclaved water that has cooled to room temperature and shake for 30 min
2. Add 8 ml 1 N NaOH
3. Add 12 ml double distilled autoclaved water

Acknowledgments

This research was supported by research grants (DEB-0322664, DEB-0423625, DEB0521177, and DEB-0228679) from the National Science Foundation as part of the Research Experience for Undergraduates and Assembling the Tree of Life Programs.
The Authors declare we have no conflicts of interest or competing interest.

References

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