Sample preparation for fPALM imaging

S. pombe labeling and fixation for fPALM imaging

Materials

• Gradient mixer – Sigma Z340391
• 3 mL syringe – Sigma Z248002
• 20 gauge needle – Sigma Z192511
• 15 ml polypropylene tube (soft) – Thermo 14-959-11B
• mCling-ATTO647N – Synaptic systems 710 006AT1
• Phalloidin-Alexa488 – Invitrogen A12379 (optional)
• NP-40 (optional)
• YE liquid media
• Phosphate-buffered saline (PBS)
• 30% lactose in PBS, filter sterilized. Keep at 37°C to prevent crystallization.
• 7% lactose in PBS, filter sterilized
• 17.4% paraformaldehyde solution (make fresh each time): 1.74 g PFA in 10 mL PBS + 40 µL 5N NaOH. Heat at 65°C for 20 min.
• 80 nm gold nanospheres – Microspheres-Nanospheres 790120. Sonicate for 1 hr to disperse clusters.
• VALAP: 1/3 vaseline, 1/3 lanolin, 1/3 paraffin. Gently heat to mix.

Lactose gradient synchronization

1. Pour a 10 mL 30%-7% lactose gradient:

• Mount the gradient mixer on a stir plate with a stir bar in the mixing chamber.
• Place the output hose into the top of a 15 mL polypropylene tube.
• With all chambers sealed, add 5 mL 30% lactose to the mixing chamber and 5 mL 7% lactose to the reservoir chamber.
• Begin stirring 
• Open the output hose valve, immediately followed by the central mixing valve to start pouring.

2. Spin down a 150 mL S. pombe culture that is ~0.3-0.5 OD₅₉₅. Resuspend in 1 mL YE and gently load on the top of the gradient.

3. Spin gradient for 7 min at 280 xg.

4. Newly divided small cells will form a hazy band near the top of the gradient. Insert syringe needle directly into this band and remove ~1-2 mL.

5. Add recovered cells to a 1.5 mL centrifuge tube. Spin down in a microcentrifuge and remove lactose supernatant.

6. Resuspend in a pre-warmed 25°C 15 mL YE culture.

7. Check cell cycle progression on a microscope for contractile rings starting at +75 min. +90 min fixation time appears perfect for capturing the highest percentage of mature contractile rings.

mCling label and fixation

1. Spin down 15 mL cultures and resuspend in 100 µL fresh YE in a 1.5 mL tube.

2. Add 1 µL 50 µM mCling-ATTO647N for 10 min.

3. Add 700 µL YE and 200 µL fresh 17.4% paraformaldehyde solution. Fix for 20 min.

4. Wash 6x with 1 mL PBS (very important! 6 washes are needed to completely remove PFA and halt fixation). Pellet cells each time at 1150 xg in a microcentrifuge. Remove supernatant after final wash.

5. Resuspend cells in 50 µL PBS with 1:100 diluted 80 nm gold nanospheres. Cells are ready for immediate imaging.

Post-fix phalloidin labeling (optional)

1. Incubate mCling-labeled and fixed cells in 3.3 µM phalloidin in PBS + 0.01% NP-40 for 30 min.

2. Wash 3x with 1 mL PBS gently. Pellet cells each time at 1150 xg in a microcentrifuge. Remove supernatant after final wash.

3. Resuspend cells in 50 µL PBS with 1:100 diluted 80 nm gold nanospheres. Cells are ready for immediate imaging.

Agar pad mounting

1. Pour a 50 µL 3% agarose pad between glass slides. Remove one slide after the agarose has solidified.

2. Add 5 µL cells in PBS + gold nanospheres to pad. Allow to slightly dry. 

3. Add coverslip, making sure no excess liquid is present (which would allow cells to move while imaging).

4. Seal on all sides with VALAP.