1. Immunofluorescence Concept
Immunofluorescence (IF) is an important immunochemical technique that can detect and localize various antigens in different types of tissues of various cellular productions.
2. Types of Immunofluorescence
Depending on the scope of the experiment or the specific antibody used, there are currently three methods: direct method, indirect method, and complement binding technique.
2.1 Direct Method
Primary or direct immunofluorescence uses a single antibody chemically linked to a fluorophore. The antibody recognizes the target molecule and binds to it, and the fluorophore it carries can be detected by microscopy. Due to the direct binding of antibodies to fluorophores, this technique has several advantages over the indirect protocol below. This reduces the steps in the staining process, making the process faster, and can reduce the background signal by avoiding antibody cross-reactivity or some problems with non-specificity. However, due to the limited number of fluorescent molecules that can bind to primary antibodies, direct immunofluorescence is less sensitive than indirect immunofluorescence.
2.2 Indirect Method
Secondary or indirect immunofluorescence uses two antibodies; Unlabeled primary antibodies specifically bind to the molecule of interest, while fluorophore-carrying secondary antibodies recognize and bind to primary antibodies. Multiple secondary antibodies can be combined into one primary antibody. This amplifies the signal by increasing the number of fluorophore molecules per antigen. This protocol is more complex and time-consuming than the direct protocol above, but it allows for greater flexibility because several different secondary antibodies and detection techniques are available for a given primary antibody.
2.3 Complement binding technique
Complement binding IIF is a very sensitive three-step serological technique. With this approach, very small amounts of circulating antibodies can be detected by their high affinity for complement. Complement can be demonstrated using FITC-labeled anticomplement. This technique is more sensitive than conventional IIF because amplification is achieved by binding more than one complement molecule to each immunoglobulin (Ig) and subsequently revealing multiple complement molecules.
3. Advantages and Disadvantages of Immunofluorescence
3.1 Advantages
(1) Simple and reproducible
(2) 1-3 hours short procedure time
(3) High sensitivity
(4) Important prognostic value, especially for herpes cases
3.2 Disadvantages
(1) Autofluorescence
(2) Fluorescence overlap
(3) Non-specific fluorescence
(4) Sometimes polymerization or affect the kinetics and expression level of target molecules
4. Application of Immunofluorescence
Immunofluorescence in autoimmune disease research; Define antigen-antibody interactions at the subcellular level; recognition of viral, protozoan, bacterial and parasitic antigens; There are many applications in areas such as identifying small cell surface structures, such as receptors on lymphocytes.
5. KMD Bioscience can Provide High-quality Immunofluorescence Technology Services
KMD Bioscience can provide high-quality immunofluorescence technology services and provide technical support. The main processes of immunofluorescence technology are as follows: specimen production, fixation, permeabilization, blocking, primary antibody incubation, secondary antibody incubation and fluorescence detection.
5.1 Specimen Production
Treat the coverslips with a 1:10 diluted polylysine solution for 5 min at room temperature to increase cell adhesion.
5.1.2 Seed cells at appropriate dilutions and grow until cells reach desired confluence (~70%)
5.1.3 Rinse the cells briefly in PBS.
5.2 Fixation
5.2.1 Aspirate PBS and cover the cells with PBS containing 2-4% formaldehyde (work in a fume hood). Allow cells to be fixed at room temperature for 15 min.
5.2.2 Aspirate the fixative and rinse 3 times for 5 min each in PBS.
5.3 Permeabilization (Optional)
Methanol and acetone fixation produce permeabilized cell productions. For paraformaldehyde fixation formulations, treat with 0.2% Triton X-100 for 5 min or with -20°C methanol for 5 min.
5.3.1 Methanol permeabilization step: After formaldehyde fixation, cover the cells with ice-cold 100% methanol (the dosage is sufficient to completely cover the cells to a depth of 3-5 mm, do not allow the cells to dry), and the cells are incubated in the methanol refrigerator for 10 min.
5.3.2 Rinse in PBS for 5 min.
5.4 Blocking
5.4.1 Block samples as secondary antibodies (e.g., normal goat serum, normal donkey serum) in PBS, 1% Triton X-5% serum in PBS, 1% Triton X-5% serum for 1 h. (The recommended blocking buffer varies between 1-2% bovine serum albumin, fetal bovine serum albumin, or 2% skim milk powder in TBS (PBS) with or without 0.2% Tween 20 and 0.02% sodium azide.) ）
Note: Unless otherwise stated, all incubations should be performed at room temperature to prevent drying and prevent exposure of fluorescent dyes to light.
5.5 Primary Antibody Incubation
5.5.1 Dilution of primary antibody in PBS, 0.1% Triton. Volume is: 50-100ul per section, 25-50ul per coverslip, chamber, or well (48- or 96-well plate).
5.5.2 Aspirate the blocking solution and apply the diluted primary antibody.
5.5.3 Incubate overnight at 4°C with gentle shaking or shaking.
5.5.4 Rinse 3 times for 5 min each in PBS and 0.1% Triton X-100.
5.6 Secondary Antibody Incubation
5.6.1 Fluorescent dye-conjugated secondary antibodies diluted in PBS, 0.1% Triton X-100 are incubated for 1-2 h at room temperature in the dark. (From this point on, the slides must be kept in the dark. ）
5.6.2 Rinse in PBS, 0.1% Triton X-100.
5.7 Fluorescence Detection
5.7.1 Install the coverslip.
5.7.2 Examine immediately using fluorescence microscopy or lay flat at 4°C in the dark.
KMD Bioscience is a reliable company providing routine and custom IHC and IF services, talented scientists with more than 10 years of experience in IHC and IF staining, and a team of experienced experts providing technical support to solve various IHC and IF technical issues to ensure the quality and speed of services.
