Immunofluorescence is a microscopy technique used to visualize the precise subcellular localization and expression levels of specific antigens (proteins, lipids, etc.) within cells or tissues by using fluorescently labeled antibodies.A primary antibody specifically binds to the target antigen. The complex is then visualized using a fluorophore-conjugated secondary antibody (indirect IF, most common) or a directly labeled primary antibody. The sample is illuminated at specific wavelengths to excite the fluorophore, which emits light of a longer wavelength, producing a detectable fluorescent signal.
Stage 1: Sample Preparation
Performed on ice or at 4°C with pre-chilled buffers.
1.1 For Cultured Cells (on Coverslips/Chamber Slides)
1.1.1 Cell Seeding: Seed cells onto sterile, poly-L-lysine or collagen-coated glass coverslips placed in a culture dish.
1.1.2 Fixation (Critical for preserving morphology and antigenicity)：
1.1.2.1 Paraformaldehyde (PFA, 4% in PBS): Most common. Fix for 10-20 minutes at RT. Mild, preserves most epitopes.
1.1.2.2 Methanol/Acetone: For certain antigens (e.g., cytoskeletal proteins). Pre-chill at -20°C. Incubate for 5-10 minutes at -20°C. Permeabilizes and fixes simultaneously. Wash 3x with PBS after fixation.
1.2 For Tissue Sections
1.2.1 Frozen Sections.
1.2.1.1 Embed tissue in OCT, section at 5-10 µm, mount on slides.
1.2.1.2 Air-dry 30-60 minutes, then fix with cold acetone or 4% PFA for 10 minutes.
1.2.2 FFPE Sections.
1.2.2.1 Follow the same deparaffinization, rehydration, and antigen retrieval steps as in IHC (Stage 1 & 2). HIER is essential.
1.2.2.2 After antigen retrieval and cooling, proceed to permeabilization.
Stage 2: Permeabilization
All steps at RT unless specified; use a humidified chamber to prevent drying.
2.1 Permeabilization (Required for intracellular antigens; optional for surface antigens).
2.1.1 Incubate samples in 0.1-0.5% Triton X-100 or 0.1% Saponin in PBS for 10-15 minutes (Saponin is milder and reversible; useful for retaining some membrane-bound structures).
2.1.2 Wash 3 x 5 minutes with PBS.
Stage 3: Blocking
3.1 Prepare a blocking buffer to suppress non-specific binding.
3.2 Common recipe: 1-5% BSA (or serum from secondary antibody host) + 0.1% Tween-20 in PBS.
3.3 Apply enough buffer to cover the sample. Incubate for 60 minutes at RT (or 37°C for 30 minutes).
3.4 Do NOT wash. Gently tap off excess blocking buffer.
Stage 4: Primary Antibody Incubation
4.1 Antibody Dilution
Dilute the primary antibody in blocking buffer to the optimal concentration (determined by titration).
4.2 Application
Apply diluted antibody onto the sample. For coverslips, place a drop (~50-100 µL) on a parafilm and invert the coverslip onto it.
4.3 Incubation
4.3.1 Option 1 (Common): 1-2 hours at RT in a humidified chamber.
4.3.2 Option 2 (Recommended for best signal/noise): Overnight at 4°C. Seal the chamber to prevent evaporation.
4.4 Washing
Carefully return coverslips to a dish. Wash 3 x 5 minutes with PBS-T (0.05% Tween-20 in PBS) with gentle agitation.
Stage 5: Secondary Antibody Incubation
5.1 Antibody Selection
5.1.1 Use fluorophore-conjugated secondary antibody raised against the host species of the primary antibody (e.g., Goat anti-Rabbit IgG).
5.1.2 Critical: Choose fluorophores with non-overlapping emission spectra for multiplexing (e.g., Alexa Fluor 488, 555, 647).
5.1.3 Dilute in blocking buffer as per manufacturer's recommendation (typically 1:500-1:2000).
5.2 Incubation
Apply the secondary antibody solution. Incubate for 60 minutes at RT in the dark.
5.3 Washing
Wash 3 x 5 minutes with PBS-T in the dark.
5.4 Optional - Nuclear Counterstain
Incubate with DAPI (4',6-diamidino-2-phenylindole), diluted 1:1000-1:5000 in PBS, for 5-10 minutes at RT in the dark. Wash 2 x 5 minutes with PBS.
Stage 6: Mounting & Imaging
6.1 Mounting
5.1.1 For slides: Drain excess PBS and apply a drop (~10-20 µL) of anti-fade mounting medium.
5.1.2 For coverslips: Place a small drop of mounting medium on a clean slide. Invert the washed coverslip (cell-side down) onto the drop.
6.1.3 Gently press to remove bubbles and seal edges with clear nail polish to prevent drying and movement.
6.2 Curing & Storage
Allow slides to cure flat in the dark for a few hours or overnight at 4°C. Store at 4°C or -20°C in the dark for long-term preservation.
6.3 Imaging
6.3.1 Image using a fluorescence or confocal microscope.
6.3.2 Use appropriate excitation/emission filters for each fluorophore.
6.3.3 Critical: Acquire images sequentially for multiplexed samples to avoid bleed-through (cross-talk) between channels.
6.3.4 Use consistent exposure times for comparative analysis.