MAMMALIAN CELLS INMUNOFLUORESCENCE (Javier Calvo)
Reagents:
1. DMEM
2. PBS
3. Fixing solution- 3-4% paraformaldehyde (in PBS)
Blocking solution-(in PBS).
0.2% powdered milk
2% NCS (Calf
serum)
0.1M Glycine
1% BSA
0.01% Triton X-100
Picks up IgGs
Blocks protein
Quenches fixing solution
Blocks non-specific
proteins
Permeabilizes cells
50 mL
100 mg
1 mL
100 mL
200 mg
2 mL
375 mg
500 mg
750 mg
1g
50 L
(of 10% stock)
100 L
(of 10% stock)
There are other blocking solutions, you can use:
1% BSA for 30minutes; 4% serum from the species that the secondary antibody was
raised; triton 0,01% for cell permeabilization.
Procedure:
1. Grow cells on sterile cover slips in the bottom of 6-24well plates until semiconfluent.
2. Aspirate media out of cells.
3. Wash twice with 1X PBS (2 mL/ well).
4. Add 3% formaldehyde (in PBS) (2 mL/well). Keep at 37C for 20 minutes (different
temperatures can be used 4ºC-37ºC)
5. Aspirate off.
6. Wash three times with 1X PBS (2 mL/ well).
7. Add 2 mL blocking solution/ well and incubate for 30 minutes at room temperature
8. Aspirate off.
9. Wash three times with 1X PBS.
10. Dilute primary antibody in filtered 0.1% BSA in 1X PBS.
11. Layer a Petri plate or bench with Para Film. Spot 200-25 L of diluted primary
antibody on Para Film.
12. With forceps, pick up the cover slip, remove excess PBS by using Kim wipe and
place cells side down on primary antibody drop. Incubate at room temperature for
60 minutes or the required time.
13. With forceps, pick up the cover slip from primary antibody spot, and place in
multiwell. Wash five times with 1X PBS (2 mL/ well).
14. Dilute secondary antibody in filtered 0.1% BSA in 1X PBS.
15. Layer another Petri plate with Para Film. Spot 200-25 L of diluted secondary
antibody on Para Film and place the coverslip over the drop for 30-45minutes on
darkness.
16. Place a drop of mounting solution(prolong or glycerol) on slides (2,5ul for small
coverslip 7-10ul for big coverslip).
17. With forceps, pick up the cover slip from secondary antibody spot, place in
multiwell and wash with PBS three times, remove excess PBS by using Kim wipe
and place on mounting solution cells side down.
18. If DAPI is used, after secondary antibody washes incubate cover slip for five
minutes in darkness and wash three times before place over the mounting media.
Antibodies drop and mounting media volume depends on the coverslip size, incubation
time and antibody dilution depends on the target protein.
For permeabilization you can use methanol -20ºC for 2minutes after fixation with
paraformaldehyde 3-4% and the three PBS washes.
If necessary a long antibody incubation use a wet chamber over the coverslip with a
Dictyostelium box and wet trapicel.
Paraformadehide preparation: It is convinient to prepare fresh each time.
Prepare 10 ml PBS (with 0,3 g PF). Place in a small beaker with a magnetic stirrer and
heat while stirring at 60-65 ºC in a gas chamber. drop NaoH to increase the ph and
allow solubilization (aprox 5 l NaOH 1M). Check that pH is not above 8. After
solubilization keep in ice until use.