- Proteins and Peptides
- Lysates and Cell Lines
Day 1 Cell Growth
1. Grow cells directly on a 10-well microscope slide (Cel-Line Associates, 10-well, 7MM, HTC autoclavable slides, Catalog# 10-7) in 15 cm cell culture plates, overnight.
- The flatter the cells, the better the IF.
- HeLa JW36 cells are a better positive control than HeLa S3 cells.
Day 2 Fixation
2. Rinse slides 3x with PBS (swirl ~200 ml into a 250 ml beaker).
3. Fix slide in Coplin jar for 30 minutes at RT, in 2% formaldehyde (MeOH-free, 10% ultrapure, EM grade).
- Formaldehyde is diluted in PBS.
4. Rinse slides 3x with PBS (swirl 200 ml into a 250 ml beaker).
5. Incubate for 3 minutes in acetone at 20C (acetone stock should be kept at 20 degrees C).
6. Rinse slides 3x with PBS (swirl 200 ml into a 250 ml beaker).
7. Store fixed slides up to 1 week in PBS + 0.2% azide, in Coplin jar.
Same Day Immunofluorescence
8. Rinse slide 1x in PBS.
9. Dry with Kimwipe (or equivalent) and wipe around well with a cotton-tipped applicator.
**This drying procedure is repeated at each subsequent step, but be careful not to dry out the well
10. Add 10 ul of 3% BSA in PBS, per well, and pre-incubate for 15 minutes at RT in a humidifier chamber.
11. Wash slide 1x in PBS, add 10 ul of anti-RNP M1-M4 and incubate 60 minutes in chamber at RT.
12. Rinse slides 3x with PBS.
13. Add 10 ul of appropriate secondary antibody and incubate for 30 minutes at RT.
14. Rinse slides 3x in PBS.
15. Mount slides with IF mounting media and seal with clear nail polish.
16. Slides can be stored up to 1 week at 20C, in Revco box.