Immunofluorescence

Microphotograph of a histological section of human skin prepared for direct immunofluorescence using an anti-IgA antibody. The skin is from a patient with Henoch-Schonlein purpura : IgA deposits are found in the walls of small superficial capillaries (yellow arrows). The pale wavy green area on top is the epidermis, the bottom fibrous area is the dermis.

Microphotograph of a histological section of human skin prepared for direct immunofluorescence using an anti-IgG antibody. The skin is from a patient with systemic lupus erythematosus and shows IgG deposit at two different places : the first is a band-like deposit along the epidermal basement membrane ("lupus band test" is positive), the second is within the nuclei of the epidermal cells (anti-nuclear antibodies).

Immunofluorescence is the labeling of antibodies or antigens with fluorescent dyes. This technique is often used to visualize the subcellular distribution of biomolecules of interest. Immunofluorescent-labeled tissue sections or cultures are studied using a fluorescence microscope or by confocal microscopy.

Uses

Most commonly, immunofluorescence employs two sets of antibodies: a primary antibody is used against the antigen of interest; a subsequent, secondary ("indirect"), dye-coupled antibody is introduced that recognizes the primary antibody. In this fashion the researcher may create several primary antibodies that recognize various antigens, but, because they all share a common constant region, may be recognized by a single dye-coupled antibody. Typically this is done by using antibodies made in different species. For example, a researcher might create antibodies in a goat that recognize several antigens, and then employ dye-coupled rabbit antibodies that recognize the goat antibody constant region (denoted rabbit anti-goat). This allows re-use of the difficult-to-make dye-coupled antibodies in multiple experiments.

In some cases, it is advantageous to use primary antibodies directly labelled with a fluorophore. This direct labelling decreases the number of steps in the staining procedure and, more importantly, often avoids cross-reactivity and high background problems. Fluorescent labelling can be performed in less than one hour with readily available labeling kits.

As with most fluorescence techniques, a significant problem with immunofluorescence is photobleaching. Loss of activity caused by photobleaching can be controlled by reducing the intensity or time-span of light exposure, by increasing the concentration of fluorophores, or by employing more robust fluorophores that are less prone to bleaching (e.g. Alexa Fluors or DyLight Fluors).

Many uses of immunofluorescence have been outmoded by the development of recombinant proteins containing fluorescent protein domains, e.g. green fluorescent protein (GFP). Use of such "tagged" proteins allows much better localization and less disruption of protein function.

Protocol

Coverslip preparation

• Coat sterile coverslip with either Gelatine (0,1% in dH2O) or polyaminoacid poly-ornithine (0,1mg/ml; e.g. for Cerebella primary cell culture)poly-D-Lysine (0,1mg/ml, e.g. Cortex). Coat with polyaminoacid either in bulk or in plate

• Wash coverslips with dH₂O or PBS if coating with polyaminoacid and let dry!
• If necessary coat coverslips with Fibronectin (e.g. bovine plasma, Calbiochem) 50µg/ml in DMEM w/o serum, diluted from 1mg/ml stock 45min at RT, gently rocking; wash 1x with DMEM
• Seed cells in appropriate density the day before transfection (e.g. 1,5x105c/6well for HeLa; 1-1,5x105c/6well for NIH-3T3)

Transfection

• If needed, transfect cells using Exgen (1- max 3µg pDNA / 6well well; 8 equivalents = 13µl/well for 3µg DNA in 100µl 150mM NaCL/1ml +DMEM or 200µl NaCL/2ml +DMEM; spin 280rcf, 5min at RT; change media after 4h til ON incubation)
• Wash cells 2x in either PBS or TBS

Fixation

• Fixation of cells using 4% PFA (Formaldehyde) in PBS/TBS or CBS (Cytoskeleton Buffer with Sucrose; if staining cytoskeletal filaments) for 5-20min at RT; or using 100% -20°C Methanol 5-20min at RT (Methanol will lead to denaturing of the protein)
• Wash cells 2x with PBS/TBS

Staining

• For Immunostaining permeabilize the cells with 0.2-0.5% Triton-X 100) in TBS 20min @ RT (harsh) or 0.05-0.1% Saponin in PBS/TBS 20min at RT (mild)
• Rinse with TBS-0,1%TX
• Block cells with either in Abdil (Antibody Dilution Solution 2% BSA) 10min @ RT or 5-10% Goat serum (in TBS) 60min at RT; or Serum appropriate for secondary antibody (ideally serum of 2nd AB producing host).
• Incubate with 1st AB overnight at 4°C in Abdil (or PBS/0.1% Triton-X 100) (e.g. 1:50-1:1000). If labelling with more than one AB apply all together (if that does not work, one after another). Pipette 50-100µl directly onto cover slip and place piece of Parafilm on top; seal plate with Parafilm
• Wash 3x 10min with TBS-0.1%TX
• Incubate with 2nd AB in Abdil (or PBS/0,1% Triton-X 100 1h at RT) (e.g. αRabbit-Cy3 1:200; Alexa-Flour-Dyes work well 1:500)
• Wash 3x 10min with PBS-0.1%TX (if counterstaining with Phalloidin or DAPI, proceed with staining after 2nd wash step)

External links

• Direct Immunofluorescence Lab Services at CTA Lab
• Images associated with autoimmune diseases at University of Birmingham
• Immunofluorescence protocol and confocal microscopy resources at confocal-microscopy.org
• Immunofluorescence Staining Protocol
• Overview at Davidson College
• Immunofluorescence at the U.S. National Library of Medicine Medical Subject Headings (MeSH)