Immunocytochemistry and immunofluorescence protocol

Immunocytochemistry

and

immunofluorescence

protocol

Procedure for staining of cell cultures using

immunofluorescence

ICC and IF protocol

Preparing the slide

1. Coat coverslips with polyethylineimine or poly-L-lysine for 1 h at room temperature.

2. Rinse coverslips well with sterile H

O (three times 1 h each).

3. Allow coverslips to dry completely and sterilize them under UV light for at least 4 h.

4. Grow cells on glass coverslips or prepare cytospin or smear preparation.

5. Rinse briefly in phosphate-buffered saline (PBS).

For wash buffer we recommend 1x PBS 0.1% Tween 20.

Fixation

The cells may be fixed using one of two methods:

1. Incubating the cells in 100% methanol (chilled at -20°C) at room temperature for 5

min

2. Using 4% paraformaldehyde in PBS pH 7.4 for 10 min at room temperature

The cells should be washed three times with ice-cold PBS.

Antigen retrieval (optional step)

Certain antibodies work best when cells are heated in antigen retrieval buffer. Check

the product information for recommendations for each primary antibody being used.

1. Preheat the antigen retrieval buffer (100 mM Tris, 5% [w/v] urea, pH 9.5) to 95°C.

This can be done by heating the buffer in a coverglass staining jar which is placed

in a water bath at 95°C.

2. Using a small pair of broad-tipped forceps, place the coverslips carefully in the

antigen retrieval buffer in the cover glass staining jar, making note of which side of

the coverslips the cells are on.

3. Heat the coverslips at 95°C for 10 min.

4. Remove the coverslips from the antigen retrieval buffer and immerse them, with

the side containing the cells facing up, in PBS, in the 6-well tissue culture plates.

5. Wash cells in PBS three times for 5 min.

Permeabilization

If the target protein is intracellular, it is very important to permeabilize the cells.

Acetone fixed samples do not require permeabilization.

1. Incubate the samples for 10 min with PBS containing either 0.1–0.25% Triton X-100

(or 100 μM digitonin or 0.5% saponin). Triton X-100 is the most popular detergent for

improving the penetration of the antibody. However, it is not appropriate for

membrane-associated antigens since it destroys membranes.

2. The optimal percentage of Triton X-100 should be determined for each protein of

interest.

3. Wash cells in PBS three times for 5 min.

Blocking and immunostaining

1. Incubate cells with 1% BSA, 22.52 mg/mL glycine in PBST (PBS+ 0.1% Tween 20) for

30 min to block unspecific binding of the antibodies (alternative blocking solutions

are 1% gelatin or 10% serum from the species the secondary antibody was raised

in: see antibody datasheet for recommendations).

2. Incubate cells in the diluted antibody in 1% BSA in PBST in a humidified chamber

for 1 h at room temperature or overnight at 4°C.

3. Decant the solution and wash the cells three times in PBS, 5 min each wash.

4. Incubate cells with the secondary antibody in 1% BSA for 1 h at room temperature

in the dark.

5. Decant the secondary antibody solution and wash three times with PBS for 5 min

each in the dark.

ICC and IF protocol

Multicolor immunostaining

(optional step)

To examine the co-distribution of two (or more) different antigens in the same sample,

use a double immunofluorescence procedure. This can be performed either

simultaneously (in a mixture) or sequentially (one antigen after another).

Ensure you have antibodies for different species and their corresponding secondary

antibodies. For example, rabbit antibody against antigen A, mouse antibody against

antigen B. Alternatively, you can use directly conjugated primary antibodies

conjugated to different fluorophores.

Simultaneous incubation

1. Incubate cells with blocking solution for 30 min.

2. Incubate cells with both primary antibodies in 1% BSA in PBST in a humidified

chamber for 1 h at room temperature or overnight at 4°C.

3. Decant the solution and wash the cells three times in PBS, 5 min each wash.

4. Incubate cells with both secondary antibodies in 1% BSA for 1 h at room

temperature in the dark.

5. Decant the secondary antibody solution and wash three times with PBS for 5 mins

each in the dark.

Sequential incubation

1. First blocking step: incubate cells with the first blocking solution (10% serum from

the species that the secondary antibody was raised in) for 30 min at room

temperature.

2. Incubate cells with the first primary antibody in 1% BSA or 1% serum in PBST in a

humidified chamber for 1 h at room temperature or overnight at 4°C, 1% gelatin

or 1% BSA.

3. Decant the first primary antibody solution and wash the cells three times in PBS, 5

min each wash.

4. Incubate cells with first secondary antibody in 1% BSA in PBST for 1 h at room

temperature in the dark.

5. Decant the first secondary antibody solution and wash three times with PBS for 5

min each in the dark.

6. Second blocking step: incubate cells with the second serum, (10% serum from the

species that the secondary antibody was raised in) for 30 min at room

temperature in the dark.

7. Incubate cells with the second primary antibody in 1% BSA or 1% serum in PBST in a

humidified chamber in the dark for 1 h at room temperature, or overnight at 4°C.

8. Decant the second primary antibody solution and wash the cells three times in

PBS, 5 min each wash in the dark.

9. Incubate cells with second secondary antibody in 1% BSA for 1 h at room

temperature in the dark.

10. Decant the second secondary antibody solution and wash three times with PBS

for 5 min each in the dark.

If you have to detect more than two antigens, continue following steps 1–5 for the

rest of the antibodies.

Counter staining

1. Incubate cells with 0.1–1 μg/mL Hoechst stain or DAPI (DNA stain) for 1 min.

2. Rinse with PBS.

Mounting

1. Mount coverslip with a drop of mounting medium.

2. Seal coverslip with nail polish to prevent drying and movement under microscope.

3. Store in dark at -20°C or +4°C.