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Section.01 How Much Do You Know About Immunostaining?
Immunostaining is a crucial biological technique primarily used to detect specific antigens in tissue sections and obtain information about cellular structures.
Immunostaining relies on the specific binding between antibodies and their target antigens (Figure 1). Detection of the antigen-antibody complex depends on markers conjugated to the antibodies. When enzymes are used, chromogenic or chemiluminescent substrates are required. The enzymatic reaction produces a colored product that can be detected using optical microscopy. For fluorescent dyes, fluorescence signals can be measured directly with a fluorescence microscope without the need for additional substrates[1].
Figure 1. Typical workflow of immunostaining[2].
Immunostaining begins with cell or tissue preparation, where specific cell or tissue samples are fixed to preserve their structure. The samples are then incubated with a blocking buffer to prevent nonspecific binding between antigens and antibodies. Subsequently, the samples are incubated with primary antibodies that specifically bind to the target antigens, followed by secondary antibodies conjugated to fluorescent dyes or enzymes. After washing to remove excess antibodies, the samples are mounted on slides using a mounting medium. Finally, the antigen-antibody complexes are visualized using an appropriate microscope.
Immunostaining: Direct or Indirect Method?
Immunostaining primarily uses two methods: indirect immunostaining and direct immunostaining.
Indirect Method
Uses both primary and secondary antibodies.
The primary antibody specifically binds to the target antigen. These are typically produced in animals (e.g., goat, mouse, or rabbit) by injecting specific antigens.The secondary antibody is conjugated to a detectable marker (e.g., enzyme or fluorescent dye) and binds to the primary antibody.To prevent cross-reactivity, it is recommended to use secondary antibodies derived from species different from those producing the primary antibodies. Multiple secondary antibodies can interact with each primary antibody, thereby amplifying the detectable signal.
Direct Method
Uses a single antibody directly conjugated to a detectable marker (e.g., enzyme or fluorescent dye) that specifically binds to the antigen.This method requires only a single antibody incubation step, making it simpler and faster than the indirect method. However, since the direct method utilizes a single antibody, its sensitivity is lower than the indirect approach. Furthermore, its applications are limited due to the restricted availability of probe/enzyme-conjugated primary antibodies.
Figure 2. Schematic diagram of direct and indirect methods.
Both methods have their own advantages. Researchers can refer to the following table (Table 1)[1].to select the appropriate method based on their experimental objectives!
Table 1. Comparison of direct and indirect immunostaining methods.
Immunostaining: ICC, IHC, and IF
Both ICC and IHC belong to immunostaining techniques. Immunohistochemistry (IHC) is used to detect specific antigens or proteins in tissue sections and is widely applied in pathology for diagnosing diseases such as cancer and studying tissue-specific protein detection. When applied to cytological preparations, it is generally referred to as immunocytochemistry (ICC), which is suitable for various cytological samples, including cell blocks, air-dried slides, ethanol-fixed slides, direct smears, cytocentrifuge preparations, and liquid-based cytology (LBC) samples[2][3].ICC is commonly used to identify the presence of specific biomarkers in cells, study subcellular localization, and investigate in situ macromolecular interactions[1].
Immunofluorescence (IF) also utilizes the binding specificity between antibodies and antigens and is a widely used example of immunostaining. IHC typically employs various enzyme labels to detect target antigens (note: IHC can also be upgraded to multiplex fluorescent staining, mIHC). ICC visualizes target antigens under fluorescence or light microscopes by using fluorescent dye- or enzyme-conjugated antibodies. IF detects fluorophore-conjugated antibodies using fluorescence microscopy and allows for multiple fluorescence staining, enabling researchers to perform multiplex labeling. IF multiplex staining is commonly used to evaluate macromolecular co-localization[1].
Note: For multiplex staining, fluorophores with non-overlapping emission spectra must be selected to prevent signal misinterpretation caused by spectral overlap.
Table 2. Characteristics of different types of immunostaining[1].
Section.02 ICC/IF: Experimental Protocol
In previous issues, we introduced immunohistochemistry. For details, please refer to: Experimental Protocol | Immunohistochemistry (IHC) Procedure + Precautions! In this issue, let's explore how to perform ICC/IF exactly?
Step 1: Sample Preparation
Cell immunofluorescence technology is a technique based on the interaction between antibodies and markers. It involves the binding of specific antibodies to cell surface or intracellular molecules, followed by observation of the markers under a fluorescence microscope. The experimental procedure typically consists of eight parts: sample preparation, fixation, permeabilization, blocking, antibody incubation, counterstaining, mounting, and observation.
Figure 3. Comparison of Direct and Indirect Immunostaining Workflows.
Purpose: Prepare well-conditioned cell samples according to the experimental design. Cell culture is the foundation of the experiment, and the cell state directly affects the experimental results.
Steps:
Step 1: Sample Preparation
1.Prepare coverslips or confocal dishes. Coverslips should be pre-soaked in 70% ethanol. After the coverslips are completely dry, transfer them to the cell culture plate, ensuring sterile conditions throughout the process.
2.Seed cells. Plate an appropriate number of cells on the coated coverslips or dishes so that the confluency reaches 50-60% during subsequent cell fixation. If the cells are too dense or too sparse, normal cell structure may be affected.
3.Collect samples. For adherent cells, continue culturing for 12 hours until the cells are firmly attached, then collect the samples for subsequent steps. For suspension cells, cytospin preparation can be used.
Tips:
1.Whenever possible, use freshly prepared samples and confirm the expression abundance of target antigen molecules.
2.Perform all subsequent steps gently to avoid cell detachment.
3.Avoid drying out the samples during all subsequent operations.
Step 2: Fixative
Purpose: To treat cells with fixatives (such as methanol, acetone, paraformaldehyde, etc.) to denature and coagulate proteins, thereby preserving cellular morphology and structure. Simultaneously, fixatives can reduce or terminate reactions of endogenous or exogenous intracellular lytic enzymes, preventing autolysis of tissue cells and protecting antigenicity.
Prepare fixative, and incubate cells with chosen fixative
Table 3. Guide to selecting fixatives for immunostaining.
Steps:
1.Fixation: Fix cells with 4% paraformaldehyde for 10-15 minutes.
2.Washing: Wash 3 times with PBS buffer to remove residual fixative.
Tips:
1.For initial experiments, start with 4% paraformaldehyde fixation for 15 minutes. If results are suboptimal, adjust fixation time or switch to an alternative fixative.
2.Longer incubation typically increases fixation intensity but may cause epitope over-fixing. Shorter incubation risks poor epitope preservation and insufficient fixation. Optimal fixation time should be determined empirically.
Step 3:Permeabilization
Purpose: Detergents partially dissolve the cell membrane to create pores, enabling antibodies to access intracellular epitopes and bind to antigens inside the cell. (This step is optional; permeabilization disrupts the membrane, making it unsuitable for cell surface antigen detection.)
How to choose permeability agent?
Figure 4. Comparison of cell permeabilization effects.
When the target protein is located on extracellular epitopes, permeabilization is unnecessary.
2. Mild Permeabilization Agents
Targets:
Intracellular domains of membrane-integrated proteins
Cytoskeletal proteins
Proteins on the outer surface of organelle membranes
Common Agents: Digitonin, Leucoperm, Saponin, Tween 20, etc.
Recommended Concentration: 0.2-0.5% (prepared in PBS solution).
Mechanism: These agents create pores in the cell membrane to enhance permeability without disrupting overall cellular architecture.
3. Strong Permeabilization Agent
Targets:
Nuclear proteins
Proteins within membrane-bound organelles (e.g., mitochondrial proteins)
Common Agents: Triton X-100, NP-40, etc.
Recommended Concentration: 0.1-0.25% (prepared in PBS solution).
Mechanism: These agents partially dissolve organelle membranes (e.g., nuclear envelope), enabling antibodies to access and bind internal epitopes.
Protocol Steps
Add 0.1-0.25% Triton X-100 (in PBS) to cover cells. Permeabilize at room temperature for 5-10 min.
Wash 3x with PBS buffer to remove residual permeabilization agent.
Tips:
Optimize permeabilization agent concentration and incubation time for your specific samples.
Figure 5. Schematic diagram of cell membrane perforation treatment.
Stage 4: Blocking
Purpose: Components in the blocking solution bind to non-specific sites on the cell surface, preventing antibodies from non-specifically attaching to these sites.
Blocking Solution Selection:
Use serum from the same species as the secondary antibody or BSA (e.g., if using goat anti-mouse secondary antibody, select goat serum as the blocking agent).
Steps:
Apply 2-10% BSA/goat serum and incubate for 1 hour at room temperature or 37°C.
Tips:
1.Avoid serum contamination: The blocking solution must not contain serum from the host species of the primary antibody, as this may cause high background noise.
2.Prevent drying: Maintain sample humidity throughout incubation to avoid drying artifacts, which significantly increase background.
Stage 5: Antibody Incubation
Purpose: Enable antibodies to bind specifically to target protein antigens. This step determines the localization and specificity of fluorescent signals, directly impacting experimental accuracy and reliability.
This step is optional.(1)Direct Detection: Primary antibodies are directly conjugated to fluorophores.(2)Indirect Detection: Use fluorophore-labeled secondary antibodies to detect primary antibodies.
Note: While both methods have advantages and limitations, indirect detection is the most widely used approach.
Antibody Selection
Single Staining:
Primary Antibody: Can be from any host species.Secondary Antibody: Must target the host species of the primary antibody (e.g., if primary is mouse-derived, use anti-mouse secondary antibodies like goat anti-mouse or donkey anti-mouse).
Double/Multiple Staining:
Primary Antibodies: Must be from different host species.Secondary Antibodies: Should match the host species of each primary antibody.Fluorophore Selection: Use spectrally distinct fluorescent labels (e.g., green, blue, and red for triple staining) to avoid signal overlap.
Steps (Indirect Detection Example)
1. Primary Antibody Incubation
Select a primary antibody specific to your target antigen.Dilute according to the manufacturer's instructions.
Incubate with samples at 4°C overnight (12-16 h).
2. Washing
Retrieve the primary antibody working solution (optional for reuse).Wash 3x with TBST/PBST on a gentle shaker (5-10 min per wash).Adjust washing time and repetitions based on experimental conditions.
3. Secondary Antibody Incubation
Choose a fluorophore-conjugated secondary antibody compatible with the primary antibody and sample autofluorescence.Incubate for 1 h at RT (protected from light).Follow the manufacturer's recommended dilution.
4. Washing
Remove/recover the secondary antibody working solution.Wash 3x with TBST/PBST (5-10 min per wash, gentle shaking).
Tips
1.After secondary antibody incubation, protect samples from light!
2.Optimize secondary antibody concentration to avoid weak/no signal or high background.
Maintain humidity in the incubation chamber to prevent drying.
3.Use gentle shaking during washes to prevent cell detachment.
Step 6: Counterstaining
Purpose: Stain nuclei to distinguish nuclear vs. cytoplasmic/antigen localization.
Steps:
Add DAPI (1 µg/mL) to washed samples.Incubate for ~30 sec at RT (protected from light).
Step 7: Mounting
Purpose: Protect stained samples and preserve results for imaging/analysis.
Steps:
Place a drop of mounting medium on a glass slide.
Invert the coverslip (cell-side down) onto the slide.
Remove excess medium with absorbent paper.
Seal edges with nail polish to prevent movement.
Store at 4°C in the dark until imaging.
Tips:
1.Avoid bubbles by applying mounting medium slowly and adjusting the coverslip gently with forceps.
2.Mounting medium selection: Common options include buffered glycerol or commercial antifade reagents. Choose based on experimental needs.
3.Store in dark, humid conditions to prevent fluorescence quenching and sample drying.
4.Imaging timeline: Samples can be stored at -20°C/4°C for ~1 week, but immediate imaging is recommended for optimal signal.
Step 8: Observation & Analysis
Acquire images using a fluorescence/confocal microscope.
Analyze fluorescence localization and experimental results.
Let's take a look at the results of immunofluorescence (IF) in the journals of researchers.
Figure 6. Immunofluorescence diagram of subcellular structure[1].
Of course, if you don't use fluorescent dyes coupled with antibodies, then your picture is like this!
Figure 7. ICC detection of tumor marker P63.
P63 staining was performed on LBC slides, showing nuclear staining that highlights myoepithelial cells. A large sheet of cells is visible in (a), while a small cluster appears in (b). Both exhibit moderate cellular atypia, with less than 25% of cells stained. If this represents the predominant morphology in the specimen, it would be sufficient to support a malignant diagnosis.
The antibody was coupled with fluorescein FITC, goat-derived and anti-mouse IgG antibody. It can be used for ICC/IF and FC experiments in mouse background.
The antibody was coupled with fluorescein FITC, goat-derived and anti-rabbit IgG antibody. It can be used for ICC/IF and FC experiments in rabbit background.
The antibody was coupled with an anti-mouse IgG antibody labeled with TRITC and derived from goats. It can be used for ICC/IF, FC experiments in mouse background.
The antibody was coupled with an anti-rabbit IgG antibody labeled with TRITC and derived from goats. It can be used for ICC/IF, FC experiments in rabbit background.
The antibody was coupled with an anti-mouse IgG antibody from goat labeled with red fluorophore Alexa Fluor® 647. It can be used in IF-Cell, IF-Tissue experiments under the background of mouse.
The antibody was coupled with green fluorophore Alexa Fluor® 488 and goat-derived anti-mouse IgG antibody. It can be used in ICC/IF, IHC-F, FC and ELISA experiments under the background of mouse.
The antibody was coupled with green fluorophore Alexa Fluor® 488 and goat-derived anti-rabbit IgG antibody. It can be used for ICC/IF, IHC-F, IHC-P, FC and ELISA experiments in rabbit background.
