Immunocytochemistry and immunofluorescence for HA-tag staining protocol
Requirements:
1. Coverslips to grow cells (Fisherbrand coverglass Cat# 12-545-83)
2. 12 well cell culture plates (Corning Incorporated Cat # 3513 or Fisher cat# 07-200-82)
3. Paraformaldehyde (Sigma Cat# P6148)
4. Image-IT Fx Signal enhancer (Invitrogen# 136933)
5. Normal goat serum (Jackson ImmunoResearch- 005-000-121)
6. Primary antibody
7. Secondary antibody conjugated with fluorescent dye
8. Mounting medium with DAPI (Vectashield mounting medium for fluorescence with
DAPI. Vector Laboratories Cat # H-1200)
9. Slides ( Fisher brand Cat# 12-550-17)
Procedure:
1. Sterilize Circular cover slips by dipping them in absolute ethyl alcohol and flaming
2. Place each coverslip into each well of 12-well culture plate and leave it under UV light
for 30 min prior to cell seeding
3. Trypsinize the cells and count under the microscope using hemocytometer. Seed 1×105
cells (1 ml culture medium) in each well containing cover slip
4. Allow cells to attach on to the cover slip at 37 °C in presence of 5% CO2 for 24 h
5. Take the attached cells for immunostaining: Prefix the cells attached on the surface of the
cover slips for 5 min by adding 500 ul of 4% paraformaldehyde into the culture medium
(1ml) 6. Remove the prefixing medium and fix the cells with 4% paraformaldehyde for 15 min
7. Wash the paraformaldehyde fixed cells with PBS for 3 times 10 minutes each
8. Permeabilize the fixed cells with PBST (PBS+0.25% Triton X-100) for 5 min
9. Wash the cells with PBS for 3 times 10 minutes each
10. Apply 3-4 drop of Image-IT TM FX signal enhancer (sufficient volume to cover the
surface of the cover slip) and incubate for 30 min at room temperature in humid
environment
11. Wash the cells with PBS for 3 times 10 minutes each
12. Incubate the cells with blocking buffer (PBS with 5% normal goat serum) at 4°C
overnight to block unspecific binding (This step can be performed at room temperature
for 1 h)
13. Wash the cells with PBS for 3 times 10 minutes each
14. Primary antibody: Incubate the cells with primary antibody (PBS with 1% BSA +
primary antibody) at 4°C overnight (This step can be performed at room temperature for
1 h)
15. Wash the cells with PBS for 3 times 10 minutes each
16. Secondary antibody: Incubate the cells with Secondary antibody (PBS with 1% normal
goat serum + Secondary antibody) at 4°C overnight (This step can be performed at room
temperature for 1 h)
17. Wash the cells with PBS for 3 times 10 minutes each
18. Mount the cover slip in a glass slide using a mounting medium with DAPI (nuclear stain)
19. Take the pictures under fluorescent microscope 20. Apply nail polish at the edge of the cover slip and store the slides at 4°C till the nail
polish dries. Then store the slides at -20°C
NOTE:
- Add enough volume of all the solutions to cover the cover slip (500 ul)
- The procedure was carried out at RT unless otherwise specified