General considerations

Throughout the procedures described below, care should be taken not to disrupt the cell specimen by vigorous shaking, e.g. on shaking platfroms etc. This includes in particular the application of solutions, which should be added by carefully pipetting them down the edge of the vessel. If incubations are carried out overnight, 0.02% sodium azide can be added to avoid the growth of contaminating microorganisms. During all steps that involve fluorophores the sample should be protected from light by wrapping it in aluminium foil or keeping it in a light-protected container.

Permeabilisation of cells

After fixation of cell specimens it is necessary to permeabilise the cells to allow antibody and reagent penetration into the sample. The standard protocol is to incubate the samples in 0.5% Triton X-100/PBS for 10 min at room temperature. Permeabilisation with methanol or acetone can be used alternatively but it should be taken into account that it causes shrinkage of the cell samples.

Blocking

To block unreacted formaldehyde groups of aldehyde fixatives and the resulting background fluorescence, samples are incubated in 50-100 mM ammonium chloride/PBS or 0.02-1% potassium borohydride/PBS for 10 minutes. To reduce unspecific binding of the antibodies, samples are treated with 10% non-immune or irrelevant serum for 30 min and during the antibody incubation steps.

Washing steps

The washing procedures during immunofluorescence labelling are crucial. In particular the washing after fixation and antibody incubation are very important to reduce unspecific background fluorescence.

Antibody binding

Two different methods can be used, direct and indirect immunofluorescence labelling. The first procedure uses a primary antibody, or a binding protein that has been covalently coupled to a fluorophore (e.g. phalloidin conjugates for F-actin labelling). The protocol for indirect labelling involves two steps, the incubation with a primary antibody raised against the epitope to be detected and a secondary antibody that is conjugated to a fluorophore and specifically recognises the primary antibody. The advantage of indirect detection is the greater flexibility in combining different secondary anitbodies with the same primary antibody in different experimental approaches. It also introduces an amplification step, because usually more than one molecule of the secondary antibody can bind each moelcule of the first antibody.

Primary antibodies - The quality of the immunofluorescence labelling is primarily dependent on the suitability of the primary antibody. Recommended are antibodies successfully tested for immunofluorescence labelling. Before carrying out an imaging experiment, the suitability and optimal dilution for each antibody should be determined. If the experiment involves the synchronous labelling of several proteins or components, primary antibodies raised in different species must be chosen. In-house raised primary antibodies should be carefully analysed by Western blotting before using them for immunofluorescence labelling and particular scrutiny should be on possible cross-reactivity. Primary antibodies are used at dilutions of 1:100 to 1:5000 in blocking solution (10% serum, 0.1% Tween 20 in PBS) and samples are incubated for 1-2 hours at room temperature under gentle agitation (orbital platform is recommended). A large range of commercially anitbodies is available from several suppliers (see Antibody Resource page). After the antibody binding, samples are washed at least 3 times for a minimum of 45 minutes in 0.1% Tween 20/PBS to completely remove unbound antibodies.

Secondary antibodies - For indirect immunofluorescence labelling the samples are incubated with secondary antibodies conjugated to sutiable fluorophores that can be detected and spectrally separated by fluorescence micriscopy. To avoid cross-reactivity, it is recommended to use F(ab) antibody fragment conjugates raised against the IgG species of the primary antibody. If more than one fluorophore is used, overlap of the different emission spectra should be avoided to allow efficient spectral separation. The Spectraviewer from Molecular Probes/Invitrogen facilitates the choice of fluorophores for multi-channel experiments. In our experience the range of AlexaFluor-labelled secondary antibodies has provided excellent results.

Epitope retrieval

If it is unavoidable to use a primary antibody that does not work optimally for immunofluorescence labelling because it was raised against a peptide that resembles more a denatured epitope, an epitope retrieval technique might be an option to improve the labelling. This technique is based on an additional step that denatures part of the cross-linked proteins in the sample by microwave heating and/or incubation with an acidic buffer. Details and protocols can be found at IHC World.