Immunocytochemistry protocol

Last edited Thu 24 Feb 2022

Immunocytochemistry (ICC) (also known as immunofluorescence (IF)) is a routine but powerful technique for visualizing the localization and distribution of your proteins of interest within cultured cells. This protocol outlines each step, including blocking with BSA, washing using ice-cold PBS, permeabilization with reagents like Triton X-100, primary and secondary antibody staining, and counterstaining. It also covers more advanced applications such as double immunofluorescence co-staining and strategies like pre-adsorbing secondary antibodies to minimize cross-reactivity. Use this cell immunostaining protocol for fluorescent visualization of your proteins of interest to aid in your research in understanding protein localization, protein function, and interactions. This guide can be adapted for several applications, from cell surface immunofluorescence staining to whole-cell imaging, both for immunofluorescence double staining or single-label experiments; use this protocol to achieve high-quality, reproducible imaging in your research.

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.

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

Prepare coating solution and filter- sterilize if necessary

Coat coverslips or plates with coating solution

• Incubation times are typically between 1 h and 24 h at room temperature.

Rinse coverslips three times with sterile PBS

Allow coverslips to dry completely. If required, sterilize them under UV light for at least 4 h

Culture cells upon coated coverslips or plates

Determine the total cell number, and check cell viability

• In general, viability should be 90 – 95%.

Prepare fixative, and incubate cells with chosen fixative

Wash cells three times with wash buffer

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 x 75 mm2 Falcon tubes/96 well plate (or any container compatible with your centrifuge)
• Suspension/washing buffer (example PBS)

Steps

Harvest and wash cells according to the manufacturer’s guidance.

Determine the total cell number, and check cell viability.

• In general, viability should be 90 – 95%.

Spin down and resuspend cells samples in ice-cold suspension buffer.

• Centrifuge at ~ 200 x g for 5 mins at 4°C.

Prepare fixative, and incubate cells with chosen fixative.

Wash cells three times with wash buffer.

• Spin down cells to a pellet (200 x g, 5 mins, 4°C)
• Remove the supernatant and resuspend the pellet after each wash.

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, example ab2601)

Steps

Prepare permeabilization solution by diluting detergent in PBS according to the following:

• Table 1: Detergents for permeabilization

Cover the cells with permeabilization solution and incubate for 2-5 min at room temperature.

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 (example – goat ab7481; donkey ab7475)
  • BSA

• PBS (example ab270748)

• Glycine

Steps

Prepare blocking buffer

• Dissolve the chosen protein blocking agent in PBS to 2 – 10 % w/v.
• Include Glycine to a concentration of 0.1 M (optional).

Block the cells by incubating cells in the blocking buffer

• Incubate at room temperature for 1 -2 h

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.

Materials required

• Cells that have undergone relevant permeabilization/blocking steps
• Antibody dilution buffer PBS (example ab270748) or PBS-T (PBS with 0.1 % Tween-20, example ab128987)
• Wash buffer PBS (example ab270748) or PBS-T (PBS with 0.1 % Tween-20, example ab128987)
• Primary antibody
• Conjugated secondary antibody
• Hydrophobic barrier pen (example ab2601)- optional for small cell volumes on slides or coverslips

Steps

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.

• If performing double immunofluorescence staining, dilute the two primary antibodies raised in different species (eg rabbit against human target-1 and mouse against human target-2, if the targets are human proteins) into one mixture.

Incubate the samples in the pre-diluted primary antibody.

• Incubate for 2 h at room temperature, or overnight at 4°C

Wash the slides three times with PBS or PBS-T.

Prepare secondary antibody in antibody dilution buffer and immerse the samples in the pre-diluted secondary antibody.

• If performing double immunofluorescence staining, dilute the two secondary antibodies raised in different species (eg rabbit against human target-1 and mouse against human target-2, if the targets are human proteins) into one mixture.
• Incubate for 1 h at room temperature.

Wash the samples three times with PBS or PBS-T

Proceed to counterstaining, mounting, and imaging

Materials required

• Cells that have undergone relevant permeabilization/blocking steps
• Antibody dilution buffer PBS (example ab270748) or PBS-T (PBS with 0.1 % Tween-20, example ab128987)
• Wash buffer PBS (example ab270748) or PBS-T (PBS with 0.1 % Tween-20, example ab128987)
• Conjugated primary antibody
• Hydrophobic barrier pen (example ab2601)- optional for small cell volumes on slides or coverslips

Steps

Determine the optimal antibody dilutions to use. Then dilute the antibodies in the antibody dilution buffer.

• If performing double immunofluorescence staining, dilute the two antibodies raised in different species (eg rabbit against human target-1 and mouse against human target-2, if the targets are human proteins) into one mixture.

Immerse the samples in the pre-diluted primary antibody.

• Incubate for 2 h at room temperature, or overnight at 4°C.

Wash the samples three times with PBS or PBS-T.

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 (ab228549), HOECHST 33342 (ab228551), or DRAQ7 (ab109202). There are also some organelle or cytoskeletal counterstains available (ab112124).

Materials required

• Cells stained with fluorophore-conjugated antibody
• Fluorescent counterstain (optional, example: DAPI ab228549)
• Mounting medium suitable for fluorescent detection (for cells on coverslips or slides)
• Sealant (for cells on coverslips or slides -example: nail polish or Limonene ab104141)
• PBS (for cells in wells, example ab270748)
• Fluorescence microscope

Steps

Immerse the samples in counterstain solution if desired.

• Incubate according to manufacturer’s guidance at room temperature, or until the desired color is observed.

Mount your samples.

Image the samples using appropriate excitation/emission filter sets and/or lasers.