Immunocytochemistry and for HA-tag staining protocol

Requirements:

1. Coverslips to grow cells (Fisherbrand coverglass Cat# 12-545-83)

2. 12 well 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 : 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