Immunofluorescence (IF) Protocol

Instructions

Immunofluorescence (IF) combines antibody-antigen reactions and fluorescence imaging to detect target proteins in cells or tissues. IF reveals the localization and relative expression of the target protein by providing image and data simultaneously.

IF diagram courtesy of BioTek. Click to enlarge.

Solution and reagents

• 4% formaldehyde
• 0.2% TritonX-100
• 10% Normal goat serum
• Primary antibody
• Secondary-fluorescence antibody
• DAPI
• PBS

Sample preparation

Cell samples

Detect the fluorescence signal with a fluorescence microscope. Cells should be cultured on a support material, such as glass coverslips or glass bottomcell culture dishes. Cell culture should be evenly dispersed and not too dense.

Tissue samples

Paraffin sections require dewaxing, hydration, and antigen retrieval steps (refer to IHC protocol), followed by incubation with antibodies to detect fluorescence. Frozen sections should first be thawed to RT (~10-20 min). Next, the sections should be fixed for 10-15min by 4% formaldehyde. Permeabilization can be performed by 0.2% TritonX-100 for 20min (also can be used to prepare cell samples).

Fixation and permeabilization

Fixation and permeabilization of samples are critical steps that can affect experimental success. Aldehyde-based fixatives (e.g., formaldehyde, formalin, glutaraldehyde) and dehydrating fixatives (e.g., ethanol) are often used. Aldehyde-based fixatives cross the membrane of cells and crosslink cellular proteins, which stabilizes the sample. Aldehyde-based fixatives tend to work better than the dehydrating fixatives, so 4% formaldehyde is recommended for cell fixation. However, some targets can lose their antigenicity when aldehyde crosslinking is used.

1. Permeabilization should be performed so that antibodies can access target proteins. Because detergents disrupt membrane structures and create pores, detergents can be used as a permeabilization reagent. Using TritonX-100 is therefore recommended. Wash cells with PBS for three times.
2. Fix the cells with 4% formaldehyde for 10-15 min at RT
3. Wash cells with PBS for three times.
4. Permeabilize the cells with 1% Triton X-100 for 20min at RT.
5. Wash cells with PBS for three times.

Blocking

Blocking reduces non-specific binding between antibodies and other proteins that are not specific to the antibody. The recommended blocking solution is normal goat serum (note: the species must be consistent with the secondary antibody). The serum not only blocks non-specific binding proteins, but it also blocks cell/tissue Fc receptors.

1. Perform blocking with 10% normal goat serum for 1 hour at RT.
2. Wipe off excess liquid and keep sample moist.

Antibody incubation

1. Prepare primary antibody solution by referring to the recommended manufacturer dilution ratio. Add enough primary antibody solution to cover sample.
2. Incubate sample with primary antibody overnight at 4℃.
3. The next day, wash cells with PBS for three times.
4. Prepare secondary antibody solution by referring to the recommended manufacturer dilution ratio. Add enough secondary antibody solution to cover sample. Keep reaction away from light.
5. Incubate sample with secondary antibody for 1 hour at RT.
6. Wash cells with PBS for three times.

Nuclear staining

DAPI is used as a nuclear reagent to stain cell structure. Dilute the DAPI reagent according to the manufacturer instructions. Incubate DAPI with the sample for the specified amount of time. Keep reaction away from light.

Detection

Protect the sample from light, and measure fluorescence signal as soon as possible.