Immunocytochemistry (ICC) protocol
Licia Miller
Product Manager
Stage 1 - Sample preparation and fixation
Fixation of the sample is an essential step to maintain cell morphology during the ICC experiment and during storage.
1, For cells cultured on coverslips
In this procedure, cells are cultured directly on coverslips before fixation.
Materials required
− Standard coverslips or multi-well plate
− Sterile PBS
− Protein for coating (e.g. Poly-L-lysine)
− Sterile water
Steps
1. Prepare coating solution and filter- sterilize if necessary.
Tips：Different sample types may require different proteins to aid in adhesion. Typical coating solutions are prepared in PBS and contain poly-L-lysine (PLL), poly-D-lysine (PDL), or gelatine. Consult the literature for your cell type of interest and recommended concentrations of your coating protein.
2. Coat coverslips or plates with coating solution.
Incubation times are typically between 1 h and 24 h at room temperature.
Tips: Place coverslips in wells of a tissue culture plate, and cover with coating liquid.
3. Rinse coverslips three times with sterile PBS.
Tips : This is to ensure the removal of free protein on the coverslip.
4. Allow coverslips to dry completely. If required, sterilize them under UV light for at least 4 h.
Warning: Alternatively, coverslips can be washed in 70% Ethanol prior to coating, and sterile solutions used throughout.
5. Culture cells upon coated coverslips or plates.
6. Determine the total cell number, and check cell viability.
In general, viability should be 90 - 95%.
7. Prepare fixative, and incubate cells with chosen fixative.
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Tips: Ethanol, methanol, and acetone will permeabilize cells so permeabilization is often not required for these fixatives. Longer incubation times generally lead to a higher degree of fixation, up to a point where epitopes may get over-fixed. Short incubation times may lead to poor epitope preservation and under-fixed samples. The optimal fixation time needs to be determined empirically.
8. Wash cells three times with wash buffer.
Tips: Ideally, the staining is performed as soon as possible after fixation. But samples may be kept in 0.1% Sodium azide/PBS for storage at 4°C for 1-2 weeks. Longer storage times may slowly reverse fixation resulting in inferior morphology and poor epitope preservation.
For suspension cells
For suspension cells, the cells can be washed and fixed before culturing the suspension onto a glass slide.
Materials required
− Cell suspension
− Polystyrene round bottom 12 × 75 mm2 Falcon tubes/96 well plate (or any container compatible with your centrifuge)
− Suspension/washing buffer (example PBS)
Steps
1. Harvest and wash cells according to the manufacturer’s guidance.
2. Determine the total cell number, and check cell viability.
In general, viability should be 90 - 95%.
3. Spin down and resuspend cells samples in ice-cold suspension buffer.
3.1. Centrifuge at ~ 200 x g for 5 mins at 4°C.
Tips: Spin times and speeds may require optimization.
4. Prepare fixative, and incubate cells with chosen fixative.
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Tips: Ethanol, methanol, and acetone will permeabilize cells, so permeabilization is often not required for these fixatives. Longer incubation times generally lead to a higher degree of fixation, up to a point where epitopes may get over-fixed. Short incubation times may lead to poor epitope preservation and under-fixed samples. The optimal fixation time needs to be determined empirically.
5. Wash cells three times with wash buffer.
5.1. Spin down cells to a pellet (200 x g, 5 mins, 4°C).
5.2. Remove the supernatant and resuspend the pellet after each wash.
Tips: Ideally, the staining is performed as soon as possible after fixation. But samples may be kept in 0.1% Sodium azide/PBS for storage at 4°C for 1-2 weeks. Longer storage times may slowly reverse fixation resulting in inferior morphology and poor epitope preservation.
6. Pipette ~ 10 µL of the suspension cells onto your slide.
The optimal cell density used depends on the used cell lines and experimental requirements.
Typical densities at this point are 106 – 107 cells/mL.
Stage 2 - Permeabilization (optional)
Permeabilization will partially solubilize cell membranes which allows antibodies to reach intracellular epitopes. This is especially required if PFA has been used as a fixative. Organic solvents like methanol generally permeabilize and fix cells at the same time, so permeabilization is not strictly required when using organic solvents as a fixative.
Materials required
− Samples that have undergone relevant fixation steps
− PBS
− Detergent (see table 1)
− Hydrophobic barrier pen (optional)
Steps
Time needed 20 minutes approx
1. Prepare permeabilization solution by diluting detergent in PBS according to the following:
Table 1: Detergents for permeabilization
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Warning: Triton X-100 is the most popular detergent for improving the penetration of the antibody. However, it is often less suitable for membrane-associated antigens since it solubilizes membranes and its associated proteins. The optimal detergent will depend on the protein and its localization.
The concentration and incubation time of detergent should be optimized for the samples being used.
If samples are on a slide, you circle the cell samples with a PAP pen to help pool the reagents.
2. Cover the cells with permeabilization solution and incubate for 2-5 min at room temperature.
Tips: If samples are on a slide, you can circle the cell samples with a PAP pen to help pool the reagents.
3. Wash cells 3 times with PBS.
Stage 3 - Blocking
Blocking steps are of particular importance in ICC to prevent high background staining in images. Typical blocking agents are 2-10 % solutions of serum proteins corresponding to the host species of the secondary antibody, or BSA, which is less species dependent, but may be less efficient for blocking. The blocking solution should not contain serum of the host animal of the primary antibody as this will likely result in high background.
Materials required
− Your samples that have undergone relevant fixation and permeabilization steps
− Protein blocking agent
− Normal serum of secondary antibody host species
− BSA (example B265991)
− PBS (example P492453)
− Glycine
Steps
Time needed 2 hours approx
1. Prepare blocking buffer.
1.1. Dissolve the chosen protein blocking agent in PBS to 2 - 10 % w/v.
1.2. Include Glycine to a concentration of 0.1 M (optional).
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2. Block the cells by incubating cells in the blocking buffer.
Incubate at room temperature for 1 -2 h
Warning: If samples are on a slide, you can circle the cell samples with a PAP pen to help pool the reagents.
3. Proceed to antibody incubation.
Stage 4 - Antibody incubation
After performing the necessary blocking steps, you’re now ready to stain your cells with antibodies. There are generally two principles for this: (i) direct ICC, where primary antibodies are conjugated directly with fluorophores, and (ii) indirect ICC, where a primary antibody is detected using a suitable fluorescently labeled secondary antibody. Both methods have advantages and disadvantages, which are discussed in more detail here. Indirect ICC is the most commonly used protocol.
Multicolor ICC involves staining cells with two or more sets of antibodies to reveal the distribution or co-localization of two or more proteins of interest. Both the indirect and direct protocols given below can be adapted for multicolor ICC. If indirect ICC is used for multicolor, it is strongly advisable to use pre-adsorbed secondary antibodies. These antibodies are pre-adsorbed for other species and thereby minimizing the chance of cross-reactivity for the used secondary antibodies to primary antibodies from different species.
Indirect
Materials required
− Cells that have undergone relevant permeabilization/blocking steps
− Antibody dilution buffer PBS (example P492453) or PBS-T (PBS with 0.1 % Tween-20, example T434505、P196391)
− Wash buffer PBS (example P492453) or PBS-T (PBS with 0.1 % Tween-20, example T434505、P196391)
− Primary antibody
− Conjugated secondary antibody
− Hydrophobic barrier pen - optional for small cell volumes on slides or coverslips
Steps
Time needed 13 hours 30 minutes approx
1. Determine the optimal antibody dilutions to use. Then dilute the antibodies in antibody dilution buffer.
Optimum dilutions will often be suggested on the antibody datasheet.
Warning: You should perform dilutions to find the antibody concentration that works best.
The antibody dilution buffer may be PBS only. The dilution buffer may also contain 0.1% Tween (to reduce surface tension) and 1% BSA (as additional blocking reagent to reduce non-specific binding).
2. Incubate the samples in the pre-diluted primary antibody.
Incubate for 2 h at room temperature, or overnight at 4°C
Warning: Incubation time and temperature may need optimization.
Tip 1: The antibody solution needs to cover your samples completely.
Tip 2: Using a hydrophobic barrier pen can help contain small volumes.
3. Wash the slides three times with PBS or PBS-T.
4. Prepare secondary antibody in antibody dilution buffer and immerse the samples in the pre-diluted secondary antibody.
Incubate for 1 h at room temperature.
Tips: Incubation time and antibody concentration may need to be optimized. Typical secondary antibody dilutions are 1:1000 or 1:2000 or between 0.1-2 µg/mL. For multi-color experiments, different secondary antibodies can often be diluted and incubated together. If using a fluorophore, incubation must be in the dark to avoid photobleaching.
5. Wash the samples three times with PBS or PBS-T.
6. Proceed to counterstaining, mounting, and imaging.
Direct
Materials required
− Cells that have undergone relevant permeabilization/blocking steps
− Antibody dilution buffer PBS (example P492453) or PBS-T (PBS with 0.1 % Tween-20, example T434505、P196391)
− Wash buffer PBS (example P492453) or PBS-T (PBS with 0.1 % Tween-20, example T434505、P196391)
− Conjugated primary antibody
− Hydrophobic barrier pen - optional for small cell volumes on slides or coverslips
Steps
Time needed 12 hours 15 minutes approx
1. Determine the optimal antibody dilutions to use. Then dilute the antibodies in the antibody dilution buffer.
Tips: Optimum dilutions will often be suggested on the antibody datasheet.
If not, you may need to perform dilutions to find the antibody concentration that works best.
The antibody dilution buffer may be PBS only. We can also recommend adding 0.1% Tween (to reduce surface tension) and 1% BSA (as additional blocking reagent to reduce non-specific binding).
2. Immerse the samples in the pre-diluted primary antibody.
Incubate for 2 h at room temperature, or overnight at 4°C.
Warning: Incubation time may need optimization.
Tip 1: The antibody solution needs to cover your samples completely.
Tip 2: Using a hydrophobic barrier pen can help contain small volumes.
3. Wash the samples three times with PBS or PBS-T.
4. Complete any appropriate counterstaining following the recommended manufacturer’s protocol.
Stage 5 - Counterstaining and detection
The final step in the ICC process is to mount the samples and detect the signal. This involves adding coverslips to slides (for cells cultures on coverslips), or adding coverslips to samples already on slides, to enable imaging.
Typical counterstain for nuclei are DAPI (D598342), HOECHST 33342 (H288601), or DRAQ7 (D266297). There are also some organelle or cytoskeletal counterstains available (P287444).
Materials required
− Cells stained with fluorophore-conjugated antibody
− Fluorescent counterstain (optional, example: DAPI D598342)
− Mounting medium suitable for fluorescent detection (for cells on coverslips or slides)
− Sealant (for cells on coverslips or slides -example: nail polish)
− PBS (for cells in wells, example P492453)
− Fluorescence microscope
Steps
1. Immerse the samples in counterstain solution if desired.
1.1. Incubate according to manufacturer’s guidance at room temperature, or until the desired color is observed.
Tips: Some mounting media are fortified with a fluorescent counterstain (see H288601). This eliminates the need to counterstain the slide separately to adding mounting medium.
2. Mount your samples.
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Tips: Mounting media often contain anti-quenchers which help to preserve fluorescence for longer.
3. Image the samples using appropriate excitation/emission filter sets and/or lasers.
For more product details, please visit the Aladdin Scientific website.