pds1::HA Immunofluorescence

Cultures: 1500, 1501
Fixations: 10, 20, 30 and 45 minutes in 4% formaldehyde, 0.1 M KPO₄ pH 6.5; 15 minutes in 4% formaldehyde, 50 mM KPO₄ pH 6.5, 0.5 mM MgCl₂

1. Grow cells to OD 0.5
2. Harvest 24 and 6 mls of each culture separately. Resuspend the '24 mls' in 10 ml 4% formaldehyde, 0.1 M KPO₄ pH 6.5, and the '6 mls' in 2.5 ml 4% formaldehyde, 50 mM KPO₄ pH 6.5, 0.5 mM MgCl₂. Put on rotating wheel at 23°C.
3. At indicated times (-2 minutes for first spin) remove 1.5 mls and wash at room temp X4 with 1 ml 0.1 M KPO₄ pH 6.5 (spin 1 min at 10K). (Can keep on ice)
4. Wash once with 0.1 M KPO₄, 1.2 M sorbitol. Resuspend in 1 ml 0.1 M KPO₄, 1.2 M sorbitol
5. Usually I spheroplast 0.5 mL of cells and store rest at 4°C (just in case). To 0.5 mL of cells, add 0.5 mL of 0.1 M KPO₄, 1.2 M sorbitol containing 1 µL bME and 10 µL oxalyticase (1 mg/mL stock).
   oIncubate at 23°C wheel; check after 20 min. Cell are spheroplasted when they swell up (explode) when water is added from the side.
6. While cells are spheroplasting, prepare polylysine coated slides .
7. Harvest spheroplasts by spinning 3-5 min at 3K. Wash once with 1.5 mL 0.1 M KPO₄, 1.2 M sorbitol, using a P1000 to gently resuspend cells--NO VORTEXING. Resuspend washed cells in 0.5 mL 0.1 M KPO₄, 1.2 M sorbitol.
8. Put 20 µL cells in each polylysine-coated well. Let cells settle 10-20 min. Aspirate sups and immediately (but gently) plunge into -20°C Methanol for 6 min. Transfer to -20°C Acetone for 30 seconds.
9. Allow slides to air dry for 1-2 min. Put 20 µL PBS-BSA (1X PBS, 1% BSA)on each well. Put slide in humid chamber for at least 5 min.
10. Aspirate PBS-BSA right before adding primary antibody.
    a) Dilute primary antibody in PBS-BSA. Try 1:1000, 1:2000, and 1:3000 for 12CA5.
    b) Use 15 µL aliquots per well.
11. Incubate at 15°C
12. Wash wells 3X with PBS-BSA. Apply secondary antibody, (Goat anti Mouse Cy3) 1:1000 . Incubate at R.T. (in the dark) for 2 hr.
13. Wash wells 3X with PBS-BSA.
14. Wash 2X with plain PBS.
15. Aspirate last wash.
16. Put a drop a mounting medium containing DAPI on each well. Put on cover slip, avoiding bubbles, and seal with nail polish. Store slides at -20°C.