Antibodies in immunofluorescence: top tips

If you work with antibodies, the chances are that you’re familiar with immunofluorescence (IF), or at least the application term. This common laboratory technique uses antibodies labelled with a fluorescent dye to detect a target antigen. Popular due to its reliability and relative simplicity, IF has a wide range of uses.

Put very simply, the labelled antibodies bind either directly (primary IF) or indirectly (secondary IF, where the second antibody carries the fluorophore) to the target antigen. Using short-wavelength, high energy light, the fluorescence can be visualised with fluorescence microscopy. This allows researchers to assess both the presence and the distribution of the target antigen in their sample.

Two particular types of IF that are often used are immunocytochemistry (ICC), the staining of cultured cells or cells that have been isolated from tissue, and immunohistochemistry (IHC), the staining of thin sections of tissue.

Whether you are new to the laboratory or a seasoned user of IF, here are the answers to a few common IF questions to help you get the most out of your antibodies.

1. What is the best method of cell fixation?

This depends on your target antigen. There are two main types of commonly-used fixative: aldehydes (such as formaldehyde and glutaraldehyde) and organic solvents (methanol, ethanol, acetone). Aldehydes are suitable for labelling cytoskeletal and membrane-bound antigens, as well as nuclear and mitochondrial proteins. Organic solvents are generally recommended for monoclonal antibodies binding to a single epitope within internal protein structures.

2. Is permeabilisation necessary?

Depending on the method of cell fixation you have chosen, a separate permeabilization step may or may not be necessary. For example, frozen samples are best fixed with methanol: this simultaneously fixes and permeabilizes, preserving cell- and secondary protein structure. However, methanol dehydrates cells, so water-soluble and lipid cell components will be lost.

If you are using an aldehyde fixative, additional permeabilization will be needed. Incubation with a detergent such as saponin or digitonin causes the cell membrane to become permeable to antibodies.

3. Antibody specificity - how important is it?

Very! Ideally, IF should be carried out with antibodies that show high specificity for the target antigen; the higher the specificity, the better. This gives the best chance of a good signal and reduced background/non-specific staining.

When choosing a primary antibody, a good starting point is to choose an antibody that is known to perform well in IF. If possible, select an antibody that was raised against the same species as you are using in the target sample.

At St John’s Laboratory, we have a catalogue of over 30,000 antibodies to choose from, many of which have been validated in immunoflourescence applications.

4. Which controls should I use for immunofluorescence?

Negative controls should be used to check the specificity of the staining. This helps to avoid mistaking autofluorescence or non-specific binding for genuine staining.

5. What is an appropriate antibody dilution?

Normally, 1-10 ug/mL of purified antibody or 1:100 to 1:1000 of anti-serum is sufficient to produce specific staining. Antibody dilution can be altered to enhance the signal, as long as the background remains low; titration experiments should be carried out on new antibodies to identify the optimal dilution.

6. Why is cell density important?

If cell density is too high, cell structures can be deformed and there can be a higher background signal at low magnifications. If cell density is too low, it can be difficult to find a field with the optimal cell pattern.

Try to choose a number of cells that will result in about 50% cell confluence at the time of staining.

7. How can I reduce the background signal?

High background signal due to non-specific staining can impact the success of IF. A blocking serum can be used to help minimise the extent of the problem. The blocking serum should be from a different species to that in which the primary antibody was raised (it can be from the species in which the secondary antibody was produced).

8. How do I mount the sample?

Mounting the sample can be tricky, but is vital in both allowing the sample to be viewed using fluorescence microscopy and in preserving the sample. Apply a drop of mounting medium – as a guide, this should take about 30 seconds to spread once the coverslip is added. Seal the samples with nail polish to prevent drying or movement of the coverslip.

Finally, when you are ready to choose antibodies for IF, don’t forget that St John’s Antibody Validation Project allows you to try up to five trial-size samples of our primary antibodies completely free! We have around 12,000 antibodies in the project, so you are sure to find something suitable.