Protocol for Staining Primary Cilia (cell culture)

Equipment

Thermo Fisher Scientific round (12 mm diameter) cover glass (thickness No 1.5).
Thermo Fisher Scientific ColorFrostTM Plus slides made of positively charge uncoated glass.

BLOCK Solution 

NaCl 8 g
KCl 0.2 g
Na₂HPO₄ 1.44 g
KH₂PO₄ 0.24 g
Tween-20 2 ml
Fetal bovine serum 50 ml
Bring to 1 L with dH₂O, stir to dissolve and adjust pH to 7.4.
Filter sterilize and store at 4^oC.

Mowiol 4-88 2.4 g
Glycerol 6 g
H₂O 6 ml
Stir for 2hrs at room temperature. Add,
0.2 M Tris 12 ml
Heat to 50^oC for 10 min and stir overnight at room temperature.
Centrifuge the solution to remove precipitates at 5,000 x g for 15min; aliquot and freeze the supernatant at -20^oC.

Preparing cell cultures

1. Wash ColorFrost^TM Plus slides (made of positively charged glass) by dipping in acetone, air dry, and store at room temperature.
2. Clean coverglass with 100% EtOH, air-dry and place into 12-well plate.
3. Seed epithelial cells, e.g. RPE1 cells into 12-well plate. Culture till confluence. Change medium to serum free medium and continue culturing for 24hrs.

1. Aspirate medium and fill the 12-well plate with -20^oC methanol. Fix at -20^oC for 1hr.

2. Aspirate methanol and let coverglass air-dry for 20min.

3. Hydrate coverglass in 1x PBS buffer.

4. Wash slides once with BLOCK solution.

5. Dilute primary antibody (anti-primary cilia protein) at 1:100 in BLOCK solution. Add the primary antibody solution to each well of the 12 well plate (500 ul is sufficient to cover the coverglass).

6. Incubate the primary antibody at 4^oC for overnight.

7. Wash the coverglass three times with the BLOCK solution.

8. Dilute secondary antibodies (anti-rabbit/rat conjugated to FITC, TRITC or other fluorophores) at 1: 200 in BLOCK solution. Add the secondary antibody solution to each well of the 12-well plate.

9. Incubate the secondary antibody at room temperature for 2hrs.

10. Wash the coverglass three times with the BLOCK solution.

11. Take out coverglass with a pair of forceps. Add 20 µl of Mowiol solution to the cell side of the cover glass. Flip the coverglass and place it onto the glass slide, and apply a small pressure to remove any trapped air bubble.

12. Let Mowiol solution dry at room temperature for 2 hours. Store glass slides at 4^oC for no longer than 1 month.