If you have a new undergrad or grad student in the lab, I would totally recommend you to give him/her a project involved with immunofluorescene microscopy. One reason is that it never fails, and it gives you pretty pictures with little efforts.
1. Grow cells on coverslips. Then conduct the necessary experiments (transfection, infection)
In the case of transfection, you can see the transfected protein produced in 24 hours. Thus, I recommend you to do 2 plates: 1 plate for 24 hours post transfection; 1 plate for 48 hours post transfection. Also, do a mock control where there is no transfection to compare if there is upregulation of your protein.
In the case of siRNA or shRNA transfection, you can see knockdown in about 72 hours. Thus, I recommend you to do 2 plates: 1 plate for 72 hours post transfection; 1 plate for 96 hours post transfection. Also, do a mock control where there is no transfection to compare if there is upregulation of your protein.
In the case of infection (ie. Look at cellular proteins after virus infection), it is best to make 3 plates: 24hpi, 48hpi, 72hpi. 96hpi may be too extreme, and most cells should be on the edge of apoptosis.
2. After the recommended time, wash cells once with ice cold 1x PBS (or DPBS). Drain the solution by aspirating with a P20/200 tip to prevent sucking up the cells
Method 1: I derived this protocol myself. It works like a charm everytime.
3. Fix cells with ice cold methanol (you can store some in -20C first) or 4% PFA for 10 mins at room temperature.
If it is a new antibody, I would do both fixation to test. Some antibodies may not work in methanol due to the way it fixes the filamentous network.
4. Wash off fixative 4 times (5 mins/wash) at room temperature with 1x PBS
5. Permeabilize cells with 0.1% Triton-X 100 in 1x PBS for 20 mins at room temperature
6. Add 1% FBS in PBS (blocking solution) for 30 mins
7. Hybridize cells with primary antibody in 1% FBS in PBS for 1 hour at 37C or overnight (preferred)
8. Wash cells with PBS for 4 times (5 minutes/wash)
9. Hybridize cells with secondary antibody in 1% FBS in PBS for 30 mins at room temperature. Cover the plates with aluminum foil to prevent light exposure.
10. Counter stain cells with DAPI for 10 mins at 37C
11. Wash cells 5 times with PBS
12. Mount on glass slide
For methanol fixation, add 500ul cold methanol. Incubate at 4C for 10 minutes. Wash twice with PBS. Block cells by adding 25% goat serum in 0.1% saponin and incubate O/N at 4C
For paraformaldehyde fixation, add 2% paraformaldehyde. Incubate at 4C for 10 mins. Wash twice with PBS. Block cells by adding 25% goat serum in 0.1% saponin and incubate O/N at 4C. Block cells by adding 25% goat serum in 0.1% saponin and incubate O/N at 4C
4. add primary antibody in 10% goat serum with 0.1% saponin for 1 hour at RT or overnight at 4C
5. wash cells three times with PBS. Add secondary antibody in 10% goat serum with 0.1% saponin. Incubate for 1 hour at RT.
6. Wash cells 3 times in PBS
7. Follow Step 10-12 in Method 1.