Staining Choanos

From ChoanoWiki

Staining choanoflagellates for tubulin and actin

Materials (for one sample)

• P20 and P200 pipettes and pipette tips
• Poly-L-lysine coated coverslip
• Glass slide
• 100% EtOH in a spray/squirt bottle
• Petri dish
• Whatman paper
• Parafilm
• 2 mL of choanoflagellates – concentration of 10^6-10^7 cells/ml
• 2 eppendorf tubes
• ~2.5 mL of PEM (100 mM PIPES, pH 6.9, 1 mM EGTA, 0.1 mM MgSO4)
• ~2.5 mL of block solution (PEM/1%BSA/0.3%TritonX-100)
• 100 µl of diluted α-tubulin 1˚ antibody (1:100, diluted in block solution)
• 100 µl of α-mouse 2˚ antibody (1:1,000, diluted in block solution)
• 100 µl of Phalloiden solution
• ProlongGold at room temperature

Preparing the Coverslip

1. Pick up coverslip with forceps

2. Spray/squirt both sides with 100% EtOH so the coverslip is completely coated

3. Prop coverslip at an angle on a pipette tip and allow both sides to air dry completely

4. While coverslips are dry, prepare a humidified chamber by cutting a circle of Whatman paper that will line the bottom of a petridish

5. Wet the Whatman and place it in the dish

6. Lay a square of parafilm on top of the Whatman that is large enough to fit all of your coverslips with at least an inch of space on all sides between each one

7. Once coverslip(s) have dried, place onto parafilm with an inch of space between each one

8. Place the lid on the dish during all incubation periods

Cell Fixation

1. Grow cells to a density of approximately 10^6-10^7 cells/ mL

2. For each coverslip to be stained collect 2 ml of cells (1 mL/ eppi tube) and add 125 µl of 37% formaldehyde to each ml

3. Incubate at room temperature for 15 minutes

4. Spin cells for 5 minutes, 500 Xg, room temperature

5. Add 100 µl PEM* to each tube and pipette up and down a few times VERY GENTLY to resuspend - pool the 2 samples

6. **Apply sample to a poly-L-lysine coated coverslip by gently pipetting sample onto the center of the coverslip so that it forms a round pool. Incubate at least 30 minutes

7. Aspirate remaining fluid GENTLY by placing tip at one corner of the coverslip and letting it touch the edge of the pool of liquid – the suction should be as low as possible to prevent damaging collar

8. Wash with PEM* 4 times by gently pipetting 100 µl of PEM onto the coverslip and subsequently aspirating the fluid off using the same corner of the coverslip.

Staining

1. ** Apply 100 µl block solution (make fresh each time!) 30 minutes.

2. **Aspirate off block solution and apply 100 µl of diluted α-tubulin 1˚ antibody incubate 1 hour at room temperature.

3. Aspirate remaining fluid, wash 4 times with block solution

4. **Apply 100 µl of diluted α-mouse 2˚ antibody and incubate 1 hour, room temperature, in a DARK DRAWER

5. Aspirate remaining fluid, wash 4 times with block solution

6. Apply 100 µl of Phalloiden solution and incubate for 15 minutes in a DARK DRAWER

7. Aspirate remaining fluid, wash 2 times with PEM*

8. Let coverslip dry almost completely in a DARK DRAWER (about 5 or 10 minutes)

9. Add 10µl of room temperature ProlongGold to a clean, dry slide and place coverslip face down on slide – be sure to place coverslip slowly and gently.

10. Allow time for ProlongGold to equilibrate (about 30 minutes) and seal with nail polish.

*PEM or PBS can be used

**Can stop for a while at these points; for antibodies, can leave at 4˚ overnight