IIF Protocol (modifications for maintaining GFP fluorescence, 8/30/2000)
- Grow cells to OD 0.5
- Harvest 0.5-1 ml cells. Resuspend in PBS, and add PFA to final concentration of 4%. If using the regular formaldehyde, add directly 10% volume to the growing media. Incubate at room temp. For HA IF use PFA and fix for no more than 20 minutes. For spindle IF, longer fixations (1-2 hours) are better. For GFP, fix for 15-60 minutes and avoid all contact with methanol.
- After fixation, wash X2 with 1 ml 0.1 M KPO4 pH 6.5 and X2 with 0.1 M KPO4, 1.2 M sorbitol (Phos/Sorb) (spin 1 min at 10K). Resuspend in 1 ml Phos/Sorb. Cells can be store overnight at this stage. Sonicate if needed.
- Spheroplast 0.5 mL of cells and store rest at 4°C (just in case). To 0.5 ml of cells, add 1 µL bME and 10 µL oxalyticase (1 mg/mL stock). Incubate at 23°C for about 20-30 min.
- While cells are spheroplasting, prepare polylysine coated slides: add 10 µl polylysine solution (0.1% in ddH2O) to each well on slide. Incubate at room temperature for about 1 minute. Remove the polylysine the add approximately 20 µl ddH2O to each well. Remove the ddH2O from each well, repeat wash and allow to air dry.
- Harvest spheroplasts by spinning 3-5 min at 3K. Wash twice with Phos/Sorb. For Triton permeabilization, go to step # 7. For methanol aceton, go to step # 8.
- Triton: Resuspend cells in 180 µl Phos/Sorb, and add 20 µl of 10% Triton. Incubate for 1-2 min at room temp, then spin gently and wash X2 in Phos/Sorb. Resuspend in 200-500 µl Phos/Sorb, and place 10-20 µl cells on polylysine coated slides. Incubate at room temp for 20 minutes, then wash X2 in PBS/BSA (1X PBS, 1% BSA). This permeabilization step can also be done directly on the slide, in which case the washed cells from the spheroplasting stage would be put down on the coated wells, settle for 10-20 minutes, the sup would be aspirated, the cells would then be treated with 1% Triton in Phos/Sorb for 1-2 minutes, washed x2 in Phos/Sorb and x4 in PBS/BSA. Continue to step 10.
- Methanol/Acetone: 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. 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.
- .Aspirate PBS/BSA right before adding primary antibody. Dilute primary antibody in PBS-BSA and put15 µL aliquots per well.
- Incubate at 1 hour at room temp or over-night at 4°C.
- Wash wells 5X with PBS/BSA. Apply secondary antibody. If secondary antibody is anti-goat, carry out the antibody reaction in PBS. Incubate at room temp (in the dark) for 2 hr.
- Wash wells 5X with PBS/BSA.
- Aspirate last wash. Put a drop a mounting medium containing DAPI on each well. Put on cover slip, avoiding bubbles, and seal with nail polish. If looking at GFP, do not use nail polish. Store slides at -20°C.
Antibody dilutions:
Primary
Mouse anti tubulin (Sigma) 1:500
Mouse anti-HA (12CA5 and 16B12) 1:250
Rabbit anti-Pds1 (for over expressed samples) 1:10,000
Secondary
All Jackson fluorescently labeled antibodies 1:200
|
