Immunocytochemistry

Immunocytochemistry is a technique used to assess the presence of a specific protein or antigen in cells by use of a specific antibody that binds to it. The antibody allows visualization of the protein under a microscope. Immunocytochemistry is a valuable tool to study the presence and sub-cellular localization of proteins.

Required materials

Cells

• Primary Rat Cortex Neurons (Cat. No. A10840-01) or Primary Rat Hippocampus Neurons (Cat. No. A10841-01)

Media and reagents

• Neurobasal Medium (1X), liquid (Cat. No. 21103-049)
• B-27 Plus Supplement (50X) (Cat. No. A3582801)
• GlutaMAX-I Supplement (Cat. No. 35050-061)
• Trypan Blue Stain (Cat. No. 15250-061)
• Dulbecco’s Phosphate-Buffered Saline (D-PBS) (1X), liquid (with calcium and
• magnesium) (Cat. No. 14040)
• Goat serum (Cat. No. 16210-064)
• MAP2, Mouse Monoclonal Antibody (Cat. No. 13-1500)
• Rabbit anti-GFAP (Glial Fibrillary Acid Protein) (Cat. No. 08-0063)
• Alexa Fluor 488 goat anti-mouse IgG (Cat. No. A11029)
• Alexa Fluor 594 goat anti-rabbit IgG (Cat. No. A11037)
• 4’, 6-diamidino-2-phenylindole, dihydrochloride (DAPI) (Cat. No. D1306)
• ProLong Gold antifade reagent (Cat. No. P36930)
• Paraformaldehyde (4%)
• Triton®-X

Special tools

• Multi-chambered slides
• Fluorescence microscope

Treating surfaces with poly-D-lysine

Treat the multi-chambered slides used in immunocytochemistry analysis with poly-D-lysine prior to analysis, as follows:

1. Prepare a 2-mg/mL stock of poly-D-lysine in nuclease-free water. Prepare aliquots and store at –20°C.
2. Prepare a working solution of the poly-D-lysine stock from step 1 in D-PBS (with calcium and magnesium) at a concentration of 50 μg/mL.
3. Add 150 μL per cm² of poly-D-lysine in D-PBS to each chamber of a multi-chambered slide (e.g., add 150 μL per chamber for an 8-chambered slide, 300 μL per chamber for a 4-chambered slide).
4. Incubate slide at room temperature for 1 hour in a tissue-culture hood.
5. Aspirate the poly-D-lysine solution, and rinse 3 times with nuclease-free water.
   Note: Rinse thoroughly, since extra poly-D-lysine can be toxic to the cells.
6. Leave the plates uncovered in the hood until the wells are completely dry. Plates can be used when dry or can be covered with Parafilm and stored at 4°C for up to two days.

Maintaining neuronal cultures

1. Thaw cryopreserved primary rat cortex cells according to the instructions provided with the cells.
2. Plate the cells onto a multi-chambered slide that has been treated with poly-D-lysine. Seed 1 × 10⁵ cells per chamber in 500 μL of medium.
3. Incubate the slide at 37°C in a humidified atmosphere of 5% CO₂ in air.
4. After 24 hours of incubation, aspirate half of the medium from each well and replace with fresh medium. Return the slide to the incubator.
5. Feed the cells every third day by aspirating half of the medium from each well and replacing with fresh medium.

Immunocytochemistry analysis

1. Before proceeding, prepare a solution of 5% goat serum in D-PBS with calcium and magnesium. This solution will be used to coat the cells before antibody detection and to dilute the antibody. Prepare enough solution to completely coat the cells twice.
2. When you are ready to perform the immunocytochemistry procedure, aspirate the supernatant from each chamber and rinse the cells twice with D-PBS with calcium and magnesium.
3. Treat the cells with 4% paraformaldehyde for 20 minutes to fix them.
4. Rinse the cells three times with D-PBS with calcium and magnesium.
5. Permeabilize the cells with 0.3% Triton®-X (diluted in D-PBS with calcium and magnesium) for 5 minutes at room temperature.
6. Rinse the cells three times with D-PBS with calcium and magnesium.
7. Add enough 5% goat serum solution from step 1 to the cells to coat them, and incubate for 60 minutes at room temperature.
8. Remove the solution from the wells and coat the cells with primary antibody (mouse anti-MAP2, 10 μg/mL, and/or rabbit anti-GFAP, 4 μg/mL) diluted in 5% goat serum solution.
9. Incubate the coated cells at 2–8°C overnight.
10. Rinse the cells three times with D-PBS with calcium and magnesium.
11. Treat the cells with a secondary antibody (Alexa Fluor 488 goat-anti mouse (H+L), 10 μg/mL, and/or Alexa Fluor 594 goat-anti rabbit (H+L), 10 μg/mL) diluted in 5% goat serum solution.
12. Incubate for 60 minutes at room temperature.
13. Rinse the cells three times with D-PBS with calcium and magnesium.
14. Stain the cells with a DAPI solution (3 ng/mL) for 10 minutes.
15. Mount the cells with ProLong Gold Antifade Reagent and observe them under the microscope using filters for FITC, Cy5, and DAPI.

Thawed cortical neurons cultured in Neurobasal Medium supplemented with B-27 Plus Supplement and GlutaMAX-I Supplement show a >90% neuron population with a minimum number of astrocytes when stained with MAP2 antibody. Within 3–4 days in culture, the neurons display extensive neurite outgrowth that keeps on increasing as long as they are kept healthy in culture. Results vary if neurons are cultured in the presence of serum.

Figure 1. Primary rat hippocampus neurons. Immunofluorescence detection of primary neuronal cells stained with mouse anti-MAP2 antibody (green) and astrocytes stained with rabbit anti-GFAP antibody (red). Nuclei are stained with DAPI (blue).