Protocols > Meiotic staining

Materials

• Carnoy I fixative: Mix one part of glacial acetic acid with three part of absolute ethanol.
• 1% Acetocarmine: Dilute 5 g of carmine powder in 500 mL of 45% (v/v) acetic acid in a 1000 mL glass beaker. Slowly bring the liquid to boil by using an electric heater (increase the heat progressively as the liquid runs out easily with heating). Boil the mixture at low heat for 60 minutes. Allow the liquid to cool down and filter it through filter paper placed into a glass funnel. Store the acetocarmine stain in a dark bottle at +4°C.
• 70% Acetic acid

Other required materials

• Razor blade
• Superfrost microscope slides
• Coverslips: 18 x 18 mm
• Fine forceps
• Cutting needle
• Dry Ice
• Hot plate or PCR equipped with a stainless steel plate

Method

1. After storing the anthers for 2 weeks in Clarke’s fluid at +4°C, remove one anther from the fixative and transfer it into 1% acetocarmine for two minutes.
2. Place the anther on a microscope slide.
3. Cut off the tip of the anther (~2 mm) and discard it.
4. Add one drop of 70% acetic acid to the slide and gently squeeze out the meiotic cells from the perpendicularly cut anther tissue into the acetic acid.
5. Remove any visible anther tissue and mix the cells into the acetic acid.
6. Carefully place an 18 x 18 mm glass coverslip on the top of the cell mixture and very gently tap the loose coverslip by moving it a few mm to the right and the left.
7. Transfer the slide to a hot plate set to 45°C and heat it for one minute. Make sure there is enough liquid under the coverslip by adjusting it with a pipette throughout the preparation and heating process.
8. Take off the slide from the hot plate by keeping the coverslip in place. If any excess liquid remains under the coverslip at this point it need to be evaporated by leaving the slide on the bench for approx. 3-5 minutes.
9. Turn the slide upside down on a clean filter paper and press it against the paper with one thumb, by avoiding any side movement.
10. Observe the quality of the slide, especially the amount of cytoplasm around the meiotic cells, by using a phase contrast microscope. Cells with a low amount of cytoplasm are suitable for immunolabelling.
11. Snap-freeze the slide on dry ice for at least 10 minutes, then take the coverslip off with the help of a razor blade.
12. The preparation can be used for immunolabelling or can be stored at -80°C for months.
13. For immunolabelling we followed the procedure described in (Lenykó‐Thegze et al., 2021).

Lenykó‐Thegze, A., Fábián, A., Mihók, E., Makai, D., Cseh, A. and Sepsi, A. (2021) Pericentromeric chromatin reorganisation follows the initiation of recombination and coincides with early events of synapsis in cereals. Plant J., 107, 1585–1602. Available at: https://onlinelibrary.wiley.com/doi/10.1111/tpj.15391.

Sepsi, A., Fábián, A., Jäger, K., Heslop-Harrison, J.S. and Schwarzacher, T. (2018) ImmunoFISH: Simultaneous Visualisation of Proteins and DNA Sequences Gives Insight Into Meiotic Processes in Nuclei of Grasses. Front. Plant Sci., 9, 1193. Available at: https://www.frontiersin.org/article/10.3389/fpls.2018.01193/full.