Immunocytochemistry Protocols

Immunocytochemistry (ICC) Protocols for Fixed or Live Cells: Indirect and Direct Methods

Detailed cell preparation and direct and indirect ICC methods.

Immunocytochemistry (ICC) allows you to locate a specific protein within or on a cell with the aid of an antibody that recognizes a particular epitope.

 

Indirect ICC uses a secondary antibody conjugated to a reporter. This secondary antibody binds the primary antibody. Even though this indirect method requires more steps and materials compared to the direct method, it benefits from signal amplification as multiple secondary antibodies – and their reporters –bind to the primary antibody. However, indirect ICC methods can also produce more background than direct ICC methods.

 

Direct ICC uses a single primary antibody to target your protein of interest. Here, the primary antibody is conjugated to a reporter such as an ATTO fluorophore. Since the direct method doesn’t require a secondary antibody, it is quicker, cheaper, and may result in less non-specific binding. However, the signal may appear weaker than the signal from indirect ICC methods, especially with proteins that have low expression levels.

 

  1. First, determine if you need to fix your cells.
  2. Next, chose either indirect (Option A) or direct (Option B) ICC methods.

 

If you have any problems, please see our extensive troubleshooting guides.

 Method

  1. Add the ATTO-conjugated primary antibody at the appropriate dilution in ice-cold Assay Buffer.
  2. Optimize your WB protocol. Refer to our WB protocol for more information. Follow the product guidelines or previous experimental optimizations for a recommended dilution for your primary antibody. Determine the quantity of antibody required for two experiments.
  3. If your optimal antibody concentration is a 1:200 dilution, add 20 µg of antibody to a 1.5 ml Eppendorf tube containing 500 µl of PBS with 1% bovine serum albumin (BSA). Label the tube “antibody alone”.
  4. To a second identical tube, add 20 µg of antibody and 20 µg of blocking peptide to 500 µl of 1% BSA in PBS. Label the tube “+peptide”.
Note: we recommend beginning with a 1:1 ratio between the antibody and the blocking peptide. You will need to test a series of dilutions to obtain full inhibition.
  5. Rotate both tubes for 1 hour at room temperature.
  6. Transfer the contents of each Eppendorf tube to larger tubes and add 4.5 ml of PBS with 1% BSA, 0.1% Tween-20, and 0.05% NaN3 to each tube to get a final antibody dilution of 1:200.
  7. Add the contents of each tube to its respective membrane test strip for parallel experiments.
  8. Incubate both membrane strips for 2–3 hours at room temperature or overnight at 4°C with gentle agitation.
  9. Proceed with the WB protocol, ensuring that you handle both the unblocked and blocked samples in the same way.
  10. Develop your blots and compare the signal obtained in the two test strips. The band that disappears when using the blocking peptide is specifically recognized by the antibody. Other visible bands represent non-specific antibody binding.

Published customer data using Alomone Labs blocking peptide controls

WB using the Anti-P2X7 Receptor Antibody (APR-004) in the presence or absence of blocking peptides.

 

WBs show a P2X7 receptor band (left panels) in epileptic mouse and human hippocampal samples blotted in the absence of the P2X7 Receptor Blocking Peptide (BLP-PR004) (-pep7r). This band was eliminated (middle panels) when the Anti-P2X7 Receptor Antibody (APR-004) was preincubated with the P2X7 Receptor Blocking Peptide (#BLP-PR004) (+pep7r). In a separate experiment, the anti-P2X7 receptor antibody was preincubated with the P2X4 Receptor Blocking Peptide (BLP-PR002) (+pep4r; right panels). As expected, the P2X4 receptor blocking peptide did not block the anti-P2X7 receptor antibody. The blots were also probed with an antibody directed against actin, which functioned as a loading control.

Adapted from Jimenez-Pacheco, A. et al. (2016) J.
Neurosci. 36, 5920 with permission of the Society for Neuroscience.

 

ICC Protocols for Fixed Cells

Cell Perception

  1. Plate the cells in chamber slides and allow them to grow for 1–2 days in an appropriate medium. Cells need to attach strongly to the plate.



    Note: some cell lines will need a special coating, e.g., polylysine on the chamber slides, to aid in cell attachment. The specific type of coating needs to be determined empirically as it varies between chamber types and cell lines.

    ICC-PBS (pH 7.4)
    Reagent Concentration
    Na2HPO4 0.016 M
    KH2PO4 0.003 M
    NaCl 0.14 M
    ICC-PBS (pH 7.4)
    Reagent Concentration
    Na2HPO4 0.016 M
    KH2PO4 0.003 M
    NaCl 0.14 M
  2. Wash the cells 3 times with ice-cold ICC phosphate-buffered saline (ICC-PBS).


    Fixation

  3. Fix the cells by adding 1–4% paraformaldehyde (PFA) in ICC-PBS and incubate for 10 minutes at room temperature.

Note: the optimal PFA concentration depends on the cell type and you need to establish this experimentally.
  4. Wash the cells 3 times with ice-cold ICC-PBS.

    Permeabilization and Blocking

  5.  Permeabilize the cell membranes by adding Saponin Assay Buffer and incubate for 10 minutes at room temperature.

    ICC-PBS (pH 7.4)
    Reagent Concentration
    Na2HPO4 0.016 M
    KH2PO4 0.003 M
    NaCl 0.14 M
  6. Block the non-specific binding sites with 5% normal serum (NS)* in Saponin Assay Buffer for 15 minutes at room temperature.

Example Data

 

Expression of the α1B-adrenoceptor in GH3 cells. Cell surface detection of α1B-adrenoceptor in living GH3 cells. The cells were stained with Anti-α1B-Adrenergic Receptor (extracellular) Antibody (AAR-018) (1:100) followed by staining with goat anti-rabbit Alexa Fluor 488 secondary antibody (green). The cell nuclei were stained with the DNA dye Hoechst 33342 (blue).

Expression of the noradrenaline transporter (NET) in live intact rat pheochromocytoma (PC12) cells. Cell surface detection of NET in live intact rat PC12 cells. The cells were stained with Anti-Noradrenaline Transporter (extracellular) Antibody (AMT-002) (1:100) followed by staining with goat anti-rabbit Alexa Fluor 594 secondary antibody (red). The cell nuclei were visualized using the cell-permeable DNA dye Hoechst 33342 (blue).

Expression of the noradrenaline transporter (NET) in live intact rat pheochromocytoma (PC12) cells. Cell surface detection of NET in live intact rat PC12 cells. The cells were stained with Anti-Noradrenaline Transporter (extracellular) Antibody (AMT-002) (1:100) followed by staining with goat anti-rabbit Alexa Fluor 594 secondary antibody (red). The cell nuclei were visualized using the cell-permeable DNA dye Hoechst 33342 (blue).