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Immunofluorescence - Protocol & Troubleshooting

Immunofluorescence (IF) is a labeled immuno-technology for antigen localization by labeling antibodies with a fluorescent substance. IF can detect and localize various antigens in a variety of cells in different types of tissues. There are two main types, including direct immunofluorescence staining and indirect immunofluorescence staining. The difference between the two is the use of secondary antibodies or not.

For IF, the protocol mainly includes cell preparation, cell fixation, serum containment, primary antibody incubation, labeled secondary antibody incubation, and fluorescence detection. We describe the principles and basic steps of IF, including common methods, solutions and reagents, and detailed operations. The specific operations can be adjusted and varied according to the actual situation and troubleshooting to determine the most suitable method.

Solutions and Reagents

Stages Solutions and Reagents
Sample Preparation Phosphate buffer (PBS), formaldehyde fixative, acetone fixative, permeabilization buffer
Blocking Blocking buffer, PBS
Antibody Incubation Primary antibody, fluorescein secondary antibody, antibody dilution buffer, PBS
Mounting and Imaging Mounting media, PBS

Immunofluorescence Procedure

Immunodiffusion

1. Sample Preparation

Suspension-cultured cell samples can be coated directly onto microscope slides in a single layer. Treat the slides to promote cell attachment, and air-dry the slides before proceeding to fixation. Tissue samples should be rapidly dissected and quickly frozen, and the sample blocks should be cut into approximately 6-8 μm sections and affixed to the slides.

2. Sample Fixation

Cover the cells with fixatives and incubate at the appropriate temperature for 10-20 minutes. And then draw out the fixative and rinse with PBS to remove the residual solution. Different fixatives can be selected according to experimental needs. Commonly used fixatives such as methanol, acetone, paraformaldehyde, and formalin.

3. Permeabilization and Blocking

The purpose of the permeabilization treatment is to permeabilize the cell membrane so that the antibody can bind to the antigen inside the cell. Permeabilize with permeabilization buffer for 1-5 minutes at room temperature, then wash with PBS. Blocking the non-specific interaction of primary and secondary antibodies with biological samples enhances IF staining. Incubate samples with blocking agents for 30-60 minutes, such as serum, then wash with PBS.

4. Antibody Incubation

Dilute the primary antibody in the blocking buffer and spread it over the sample. The range of antibody dilution depends on the antigen abundance, the antibody concentration, and the affinity of the antibody. Samples are incubated with primary antibodies and then washed with PBS to remove unbound primary antibodies. Dilute the fluorophore-conjugated secondary antibody in the blocking buffer and add it to the sample. Samples are incubated with the secondary antibody and then washed with PBS.

5. Mounting and Imaging

Before covering the sample with a coverslip, add a small amount of mounting medium sufficient to cover the sample. The sample is then allowed to dry and the edges are sealed. After mounting, the sample can be imaged for observation. Antigens of interest can be visualized by fluorescence microscopy or confocal microscopy.

Troubleshooting

Creative Biolabs has listed some common IF troubleshooting tips. We provide you with tips covering many different problems you may encounter in IF. And we hope this information is useful to you and serves as a useful reference guide.

Weak or no fluorescence expression

  1. Sample causes. The target antigen may not be present in the sample being tested. You can run a positive control to check if the antigen is inside the cell or on the cell surface. If the antigen is present but not abundant, use an amplification step to maximize the signal. Antigens may be destroyed prior to incubation with the antibody. The target protein should be correctly induced. Determine optimal processing conditions and control conditions for the target antigen. It is important to note that the sample must remain in liquid throughout the staining process.
  2. Fixation causes. Inadequate or excessive fixation of cells/tissues can result in weakened or lost signals. You should increase/shorten fixation time as appropriate or simply choose another fixation method. If the sample is over fixed resulting in epitope damage, the antigen can be exposed by treatment with antigen repair reagents.
  3. Permeabilization causes. Poor cell permeabilization can result in staining that does not reach intracellular targets and thus no signal is observed. You can optimize the permeabilization step by increasing the concentration of permeant or the reaction time. If the antibody is lost due to permeabilization, you need to decrease the concentration or reaction time of the permeabilizing agent.
  4. Antibody causes. First, ensure the suitability of the antibody. Check the function of the antibody by using a positive control and verify that it is recommended for your application. Too low concentrations of antibodies result in too few primary antibodies available to bind to the target antigen. Higher concentrations of antibodies or polyclonal antibodies can be used to enhance the signal and incubate for longer periods of time. You can also perform antibody concentration titrations to find the optimal concentration to optimize antibody conditions. Antibodies may also be inactive and should be reported through an independent test reporting system to assess the viability of the reagent. Antibodies will not work due to improper storage. Freezing/thawing cycles of antibodies are harmful and antibodies should be stored according to instructions.
  5. Secondary antibody causes. If the primary and secondary antibodies are incompatible, little or no fluorescence will be observed. You need to make sure that the type of secondary antibody matches the type of primary antibody. This can also occur if the secondary antibody is incompatible with the serum protein used for blocking. So, you need to avoid cross-reactivity between the secondary antibody and the immunoglobulin in the blocking agent.
  6. Microscope causes. First make sure you have selected the proper microscope detection method, if not, you will need to use a more sensitive image acquisition method or use a brighter fluorophore for detection. Check dye excitation/emission settings to make sure your microscope is equipped with the correct light source and filter set for the fluorophore. You also can turn up the gain or increase the exposure time to ensure that you capture any signal present.

High background

  1. Sample causes. The sample must be kept in liquid throughout the procedure to avoid drying of the sample. Check the autofluorescence of the tissue sample. Check if sections are too thick, consider using thinner tissue sections. If permeabilization destroys the cells, the permeabilization step should be reduced or skipped.
  2. Antibody causes. If the signal and background are high, the antibody concentration may be too high, the incubation time too long, or the incubation temperature too high. You need to reduce the antibody concentration, dilute according to the recommended antibody dilution and optimize the antibody conditions. Try running the secondary antibody without the primary antibody. If there is staining, this indicates that non-specific binding has occurred. The secondary antibody should be replaced. It is also possible that the antibody and protein are interacting hydrophobically, you can reduce the ionic strength of the antibody diluent.
  3. Fixation causes. Improper or excessively long fixation can cause artifacts. Reduce fixation time or change fixative to reduce noise.
  4. Blocking causes. If a high level of background is present, it may be caused by insufficient blocking. Please consider changing the blocking solution or increasing the blocking time.
  5. Washing causes. It is critical to wash correctly between steps. Make sure you follow the protocol guidelines for the wash steps.

Fast fluorescence quenching

Too large or too small a precipitation ring can result in inaccurate ring diameter measurements.

  1. Fluorescein causes. The reason may be the poor stability of fluorescein. A fluorescein secondary antibody with good light stability can be used. Or use a sealer that prevents fluorescence burst.

We are committed to helping you achieve superior research results through the protocol. If you need more help with your experiments, please contact us for more assistance.

Reference

  1. Joshi S and Yu D. Immunofluorescence. Basic science methods for clinical researchers. Academic Press, 2017, 135-150.

For research use only. Not intended for any clinical use.

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