Immunocytochemistry/Immunofluorescence

Immunofluorescence (IF) (also known as immunocytochemistry (ICC)): Cell immunofluorescence is a technique based on the interaction between antibodies and markers. Specific antibodies bind to molecules on the cell surface or inside the cell, and the distribution of the target molecules is visualized under a fluorescence microscope using the markers. The experimental procedure typically includes eight parts: sample preparation, fixation, permeabilization, blocking, antibody incubation, nuclear counterstaining, mounting, and observation^[1].

Development and Application: Immunofluorescence technology is widely used and can be used to label various substances such as proteins, peptides, and nucleic acids. With the development of technology, confocal laser scanning microscope (CLSM) has become an important tool for immunofluorescence technology. CLSM can use laser as a scanning light source to quickly scan and image point by point, line by line, and surface by surface. Compared with ordinary fluorescence microscopes, CLSM has a higher resolution and can see the structure inside the cell; in addition, CLSM can scan different layers and display the three-dimensional structure of cell samples through computer analysis and simulation, which is an advantage that traditional fluorescence microscopes cannot match. CLSM has been used for cell morphology localization, three-dimensional structure reconstruction, dynamic change process and other research, and provides quantitative fluorescence measurement, quantitative image analysis and other research methods. Combined with other related biotechnology, it has been widely used in molecular cell biology fields such as morphology, physiology, immunology, and genetics^[2].

Immunofluorescence experiments are complex and require not only high-quality antibodies but also experienced operating skills. MCE provides you with high-quality immunofluorescence-specific reagents and professional technical support services. For more service information, please visit the official website: https://www.medchemexpress.com/

MCE has not independently verified the accuracy of these methods; they are for reference only.

1. Sample preparation

(1) Prepare coverslips or confocal dishes: Soak coverslips in 70% ethanol beforehand. After the coverslips are completely dry, transfer them to the cell culture plate. Maintain aseptic technique throughout the process.
(2) Spread cells: Spread an appropriate amount of cells onto the coated coverslips or plates to ensure 50-60% confluence for subsequent cell fixation. Overcrowding or sparse placement of cells may affect normal cell structure.
(3) Collect samples: For adherent cells, continue culturing for 12 hours until firmly attached. Collect samples for further processing. For suspension cells, this can be done by swishing the slide. (Avoid drying the slide during all subsequent operations and handle the slide gently to prevent cell detachment.)

2. Fixation

Cells are treated with fixatives (such as methanol, acetone, paraformaldehyde, etc.) to denature and coagulate proteins, thereby fixing cell morphology and structure. Simultaneously, fixatives can reduce or terminate the reactions of endogenous or exogenous intracellular degradative enzymes, preventing cell autolysis and protecting antigenicity.
(1) Fixation: Fix cells with 4% paraformaldehyde for 10-15 min. For initial experiments, it is recommended to start with 4% paraformaldehyde for 15 min. If the desired effect is not achieved, adjust the fixation time or change to another fixative. (Longer incubation times usually result in higher fixation, up to the point that epitopes may be over-fixed. Shorter incubation times may lead to poor epitope preservation and insufficient sample fixation. The optimal fixation time needs to be determined empirically.)
(2) Washing: Wash three times with PBS buffer to remove residual fixative.

3. Permeabilization

The cell membrane is partially dissolved by a permeabilizer, creating pores that allow antibodies to reach intracellular epitopes and bind to intracellular antigens. (This step is optional; permeabilization will damage the cell membrane, and cell membrane surface antigens are not suitable for permeabilization experiments.)
(1) Permeabilization: Add 0.1-0.25% Triton X-100 (prepared with PBS) to cover the cells and permeabilize at room temperature for 5-10 min. (The concentration of the permeabilizer and the incubation time should be optimized for the sample used.)
(2) Washing: Wash three times with PBS buffer to remove residual permeabilizer.

4. Blocking

The components in the blocking solution can bind to nonspecific sites on the cell surface, thereby preventing the antibody from binding nonspecifically to these sites. Serum or BSA from the same source as the secondary antibody can be selected; for example, if the secondary antibody is goat anti-mouse, goat serum should be chosen as the blocking agent. The blocking solution should not contain the host animal serum of the primary antibody, as this may lead to high background.
(1) Blocking: Use 2-10% BSA/goat serum and block at room temperature or 37°C for 1 h. (Keep the sample moist and avoid drying it, otherwise high background is very likely to occur.)

5. Antibody incubation

The binding of the antibody to the target protein antigen determines the location and specificity of the fluorescence signal, thus affecting the accuracy and reliability of the experimental results. The most commonly used detection method is indirect detection.
(1) Primary antibody incubation: Select a suitable primary antibody based on the target antigen and sample characteristics, dilute it according to the instructions, add it to the sample, and incubate overnight (12-16 h) at 4°C.
(2) Washing: Recover the primary antibody working solution and wash slowly three times with a TBST/PBST shaker for 5-10 min each time to remove unbound antibodies. (The number of washes and time can be adjusted according to experimental conditions; the washing speed should be gentle to prevent cell detachment.)
(3) Secondary antibody incubation: Select a suitable fluorescently labeled secondary antibody based on the type of primary antibody and sample characteristics (the fluorescence carried by the secondary antibody should not conflict with the fluorescence of the sample itself), and incubate at room temperature in the dark for 1 h. Dilution and usage methods should follow the instructions. (Determine the appropriate working concentration of the secondary antibody to avoid signal loss or excessively high background. Pay attention to the humidification effect of the humidifying box to prevent sample drying.)
(4) Washing: Remove/recover the working solution of the secondary antibody. Wash slowly three times with a TBST/PBST shaker for 5-10 min each time to remove unbound antibodies. (The number of washes and time can be adjusted according to experimental conditions. The washing speed should be gentle to prevent cell detachment.)

6. Nuclear Counterstaining

The cell nucleus is stained with a staining agent to visually distinguish the location of the cell nucleus from other cells and antigen structures, facilitating the observation and interpretation of experimental results.
(1) Nuclear staining: Add DAPI/Hoechst to the washed cells and let them stand at room temperature in the dark for about 30 seconds.

7. Mounting and Observation.

Mounting can protect stained samples from damage caused by the external environment. Mounting also helps preserve experimental results, facilitating subsequent analysis and comparison.
(1) Mounting: Slowly and evenly drop an appropriate amount of anti-fluorescence quenching mounting solution onto the slide. Use tweezers to gently place the coverslip onto the slide, avoiding air bubbles. The cell side should be close to the slide. Use absorbent paper to remove excess mounting material. Gently seal the coverslip with nail polish to prevent sample movement under the microscope. After the nail polish dries completely, observe directly or store at 4°C in the dark.
(2) Observation: Observe and acquire images under a fluorescence microscope or laser confocal microscope, and perform experimental analysis on fluorescence localization and results.

1. Sample selection and washing: Use freshly prepared, well-grown cells whenever possible, and determine the expression abundance of antigen molecules in the sample. Maintain sample moisture throughout the entire process to avoid drying out the slides. The washing time and number of washes can be determined based on the actual experimental conditions, and the movements should be gentle to avoid cell detachment.
2. Reagent selection, working concentration, and incubation time: The optimal working concentration and incubation time for fixatives, permeabilizers, blocking solution, and antibodies may different. Adjustments should be made promptly during actual operation to ensure that the fluorescence of the target molecule is clearly visible with minimal background noise during final observation.
3. Selection of detection method: (i) Direct detection: The primary antibody is directly coupled to the fluorescent group. The advantages of this method are simple operation, high specificity, less non-specific staining, and easy co-localization of different proteins from the same host. However, its sensitivity is relatively lower than the indirect method, and the cost is relatively higher. (ii) Indirect detection: The primary antibody is detected using a suitable fluorescently labeled secondary antibody. The advantages of this method are high sensitivity, more flexible staining, and low cost. However, it is more complex to operate than the direct method, and its specificity is slightly lower.
4. Selection of antibody: (i) Single staining: The primary antibody can be an antibody from any host, and the secondary antibody should be an antibody against the corresponding host of the primary antibody. For example, if the primary antibody is from a mouse, the secondary antibody should be an anti-mouse antibody (such as goat anti-mouse, donkey anti-mouse, etc.). (ii) Double/Multiple staining: When performing multiple fluorescently labeled experiments, the primary antibody should be an antibody from different host sources, and the secondary antibody should be an antibody against the corresponding host of the primary antibody. It is also important to select a fluorescent secondary antibody with high discriminative power to avoid fluorescence overlap. For example, when performing tricolor fluorescent labeling, green, blue, and red fluorescent groups are generally selected to ensure that each signal can be clearly distinguished. 5. Sample preservation: During and after secondary antibody incubation, the sample should be stored in a dark environment with suitable humidity. This effectively prevents fluorescence signal quenching and sample drying, ensuring the stability and accuracy of experimental results. Theoretically, mounted sample can be stored at -20°C/4°C for one week. However, for clear imaging and high fluorescence intensity, it is recommended to perform imaging analysis as soon as possible.
6. Observation order: When observing and acquiring fluorescence images of multiplexed samples, the weakest and most easily quenched channel signal should be acquired first, followed by those with strong fluorescence signals.