Immunofluorescent / Immunocytochemistry Protocol

Immunofluorescent (IF)/Immunocytochemistry (ICC) is a common laboratory technique that uses antibodies that target molecule of interest in the . IF/ICC allows researchers to evaluate whether or not cells in a particular sample express the antigen in question. It is a valuable tool for the determination of cellular contents from individual cells. Samples that can be analyzed include blood smears, aspirates, swabs, cultured cells, and cell suspensions.

Materials and Equipment Required 1.1 Reagents Formaldehyde Buffer 1 g Formaldehyde 100 ml PBS Buffer Store at 4 °C

Blocking Buffer 10 ml Normal Goat antiserum 90 ml PBST Buffer Store at 4 °C

PBST Buffer 0.5 ml Tween 20 1000 ml PBS Buffer Store at 4 °C

Antibody Dilution Buffer 1 g BSA 100 ml PBST Buffer

0.1% Triton X-100 Buffer 0.1 ml Triton X-100 100 ml PBS Buffer

PBS Buffer 8.5 g NaCl

1.4 g Na2HPO4

0.2 g NaH2PO4

1000 ml dd H20 Adjust pH to pH 7.4 Store at 4°C

Others:

Primary antibody/antibodies

Fluorophore-labeled secondary antibody/antibodies

Cells of interest

Tissue culture medium appropriate for the cells

6-well tissue culture plates

Glass coverslips

Broad- Tipped Forceps

1.2 Equipment:

Fluorescence microscope fitted with filters for: Texas Red (TXRD) dye (Excitation λ 590nm; Emission λ 620nm). Fluorescein isothiocyanate (FITC) dye (Excitation λ 488nm; Emission λ 520nm). DAPI dye (Excitation λ360nm; Emission λ 460nm).

2. Procedure 2.1 Cell preparation Seed the adherent cells on 6-well tissue culture plates in a sterile tissue culture hood. 1. Sterilize glass coverslips by dipping them in 90% ethanol and carefully dry them over a flame for a few seconds. 2. Place each coverslip in sterile 6-well tissue culture plates. 3. Add 1-2ml of cell suspension over each coverslip in the 6-well plates.

4. Grow the cells at 37 °C in a humidified CO2 incubator until they are 50-70% confluent. 5. Aspirate the culture medium from each well and gently rinse the cells twice in PBS at room temperature. Note: Do not let the cells dry out.

2.2 Fixation Note: this step is not necessary for cell surface antigens staining 1. Incubate cells with Formaldehyde Buffer and fix cells for 10 minutes at 37°C. 2. Incubate cells in PBS Buffer and then decant supernatant. Note: The cells can be stored in 0.02%(w/v) sodium azide in PBS at 4°C for several days.

2.3 Permeabilization 1. Incubate cells with ice-cold 0.1% Triton X-100 Buffer for 10 minutes on ice to permeabilize the cell membrane. 2. Rinse the cells 3 times with PBS Buffer

2.4 A. For un-conjugated primary antibody 1. Dilute the unconjugated primary antibody and the corresponding unconjugated isotype control antibody separately in Antibody Dilution Buffer. Note: The primary antibody should be diluted at the optimal dilution according to the manufacturer’s instructions 2. Incubate the cells with the diluted primary antibody or diluted isotype control antibody for 1 hour at 37°C. 3. Incubate the cells with PBS buffer for 10 minutes and then discard the supernatant. Repeat twice. 4. Dilute the corresponding fluorochrome conjugated secondary antibody in Antibody Dilution Buffer. 5. Incubate the cells with the diluted secondary antibody for 40 minutes at 37°C. Note: the secondary antibody should be diluted at the optimal dilution according to the manufacturer’s instructions and this incubation must be done in the dark. 6. Incubate the cells with PBS buffer for 10 minutes and then discard the supernatant. Repeat twice.

B. For fluorochrome conjugated primary antibody 1. Dilute the fluorochrome conjugated primary antibody and the corresponding fluorochrome conjugated isotype control antibody separately in Antibody Dilution Buffer. Note: the primary antibody should be diluted at the optimal dilution according to the manufacturer’s instructions 2. Incubate the cells with the diluted primary antibody or diluted isotype control antibody for 1 hour at 37°C. Note: this incubation must be done in the dark. 4. Incubate the cells with PBS buffer for 10 minutes and then discard the supernatant. Repeat twice. 2.5 Mount coverslips and visualize under microscope 1. Label a microscope slide for each coverslip. 2. Add a drop of mounting medium to each slide. 3. Pick up each coverslip with a forceps and place it on the mouting medium, with the cell-side face down. 4. Apply nail polish or glue along the edges of the coverslips to seal them to the slides. 5. Visualize the cells using a equipped with the appropriate filters for DAPI dye, Texas Red

dye and Fluorescein isothiocyanate dye.

3. IF/ICC Examples

Immunocytochemistry/ analysis of Strep II tagged protein transfeced HEK293 cells using THE™ NWSHPQFEK Tag Antibody, mAb, Mouse (GenScript, A01732) and Mouse IgG Control (Whole Molecule), Purified (GenScript, A01007)

The signal was developed with FITC conjugated Goat Anti-Mouse IgG.

Immunocytochemistry/Immunofluorescence analysis of GFP fusion protein transfeced HEK293 cells using THE™ GFP Antibody, pAb, Rabbit (GenScript, A01704) and Rabbit IgG Control (Whole Molecule), Purified (GenScript, A01008)

The signal was developed with R-PE conjugated Goat Anti-Rabbit IgG.

4. Recommended Products

Name Cat. No. Size Price Human IgG Control(Whole Molecule), Purified A01006 4 mg $50.00 Mouse IgG control (Whole Molecule), Purified A01007 1 mg $50.00 Rabbit IgG Control (Whole Molecule), Purified A01008 4 mg $50.00 Goat IgG Control (Whole Molecule), Purified A01009 4 mg $50.00 Chicken IgY Control (Whole Molecule), Purified A01010 4 mg $50.00

5. Troubleshooting Problem Possible Cause Solution Increase the concentration of primary Antibody concentration is and/or secondary antibodies. Weak or No staining too low Use secondary antibody that will interact

with primary antibody. Replace with a new batch of antibody Non-specific binding of primary antibodies to tissue Non-specific binding may be reduced by or antibody concentration is using high dilution of primary antibodies too high High background Use pre-adsorbed 2nd antibody, i.e. use Non-specific binding of rabbit anti-rat IgG, mouse adsorbed, on secondary antibodies to mouse tissue, or use rabbit anti-mouse tissue IgG, rat adsorbed, on rat tissue. Inadequate washing of Wash at least 3 times between steps sections

GenScript USA Inc. 860 Centennial Ave., Piscataway, NJ 08854 Tel: 1-877-436-7274, 1-732-885-9188 Fax: 732-210-0262, 732-885-5878 Email: [email protected] Web: www.genscript.com