TITLE: Immunofluorescence staining
AUTHORS: michela.deleidi, Pascale Baden, María José Pérez J., Hariam Raji, Federico   Bertoli

[DESCRIPTION]
This protocol describes the immunofluorescence staining of cells.

[STEPS]
SECTION: Fixation of the cells: Strategy 1, i.e., HEK cells
1. Remove medium.
SECTION: Fixation of the cells: Strategy 1, i.e., HEK cells
2. Wash with 1× PBS.
SECTION: Fixation of the cells: Strategy 1, i.e., HEK cells
3. Remove PBS and add 4% PFA to the cells.
 

SECTION: Fixation of the cells: Strategy 1, i.e., HEK cells
4. Incubate 10 min at Room temperature.

SECTION: Fixation of the cells: Strategy 1, i.e., HEK cells
5. Collect PFA.
SECTION: Fixation of the cells: Strategy 1, i.e., HEK cells
6. Wash 1× PBS.
SECTION: Fixation of the cells: Strategy 1, i.e., HEK cells
6.1. Wash with 1× PBS for 5 min at Room temperature. (1/3)

SECTION: Fixation of the cells: Strategy 1, i.e., HEK cells
6.2. Wash with 1× PBS for 5 min at Room temperature. (2/3)
SECTION: Fixation of the cells: Strategy 1, i.e., HEK cells
6.3. Wash with 1× PBS for 5 min at Room temperature. (3/3)
SECTION: Fixation of the cells: Strategy 2, i.e., neurons
7. Add the same volume of 8% PFA as medium is in the well to the well.
SECTION: Fixation of the cells: Strategy 2, i.e., neurons
8. Incubate 10 min at Room temperature.
SECTION: Fixation of the cells: Strategy 2, i.e., neurons
9. Collect PFA in a falcon.
SECTION: Fixation of the cells: Strategy 2, i.e., neurons
10. Wash in 1× PBS.
SECTION: Fixation of the cells: Strategy 2, i.e., neurons
10.1. Wash with 1× PBS for 5 min at Room temperature. (1/2)
SECTION: Fixation of the cells: Strategy 2, i.e., neurons
10.2. Wash with 1× PBS for 5 min at Room temperature. (2/2)
 

SECTION: Blocking and permeabilization
11. Remove 1X PBS.
SECTION: Blocking and permeabilization
12. Add 300 µL/well of blocking solution. 
 


SECTION: Blocking and permeabilization
13. Incubate at least 60 min at Room temperature.

SECTION: Staining: Day 1
14. Prepare antibody in blocking solution containing 5% NGS.
SECTION: Staining: Day 1
15. Put a drop (50 µL) of Primary antibody solution on the parafilm surface.

SECTION: Staining: Day 1
16. Remove the coverslips from the plate and gently put it upside-down on the antibody drop.
SECTION: Staining: Day 1
17. Incubate 60 min at 4 °C.

SECTION: Staining: Day 2
18. Wash: 
SECTION: Staining: Day 2
19. Prepare secondary antibody in blocking solution containing 5% NGS. 
 

SECTION: Staining: Day 2
20. Put a drop (50 µL) of secondary antibody solution on the parafilm.

SECTION: Staining: Day 2
21. Take the coverslips and put it upside-down on the antibody drop.
SECTION: Staining: Day 2
22. Incubate 60 min at Room temperature in the dark.

SECTION: Staining: Day 2
23. Transfer the coverslip to a 24-well containing 500 µL 1X PBS + Triton X-100 0,1%.

SECTION: Staining: Day 2
24. Incubate for 5 min at Room temperature (in the dark).

SECTION: Staining: Day 2
25. Dilute DAPI 1:10000 in 1X PBS.
SECTION: Staining: Day 2
26. Remove PBS and incubate with DAPI for 5 min at Room temperature in the dark.

SECTION: Staining: Day 2
27. Wash.
SECTION: Staining: Day 2
28. Mount the slides:
SECTION: Staining: Day 2
18.1. Wash for 5 min in PBS + Triton X-100 0.1%. (1/3)

SECTION: Staining: Day 2
18.2. Wash for 5 min in PBS + Triton X-100 0.1%. (2/3)
SECTION: Staining: Day 2
18.3. Wash for 5 min in PBS + Triton X-100 0.1%. (3/3)
SECTION: Staining: Day 2
27.1. Wash with 1X PBS. (1/2)
SECTION: Staining: Day 2
27.2. Wash with 1X PBS. (2/2)
SECTION: Staining: Day 2
28.1. Put a drop (10 µL) of DAKO mounting reagent on the slide.

SECTION: Staining: Day 2
28.2. Take out the coverslip from the plate.
SECTION: Staining: Day 2
28.3. Dry it by gently tapping the coverslip’s edge on a lens-cleaner tissue.
SECTION: Staining: Day 2
28.4. Gently put the coverslips upside-down on the DAKO drop.
SECTION: Staining: Day 2
28.5. Leave it dry (1440 min, DARK, Room temperature).

SECTION: Staining: Day 2
29. Store in a slide-box at 4 °C.