Practical considerations for immunofluorescent staining of macrophage populations in vitro

2.2.1 Direct versus indirect immunochemical labelling

There are two methods to detect antigens expressed by macrophages (5). Coupling of a detectable label (fluorochrome or enzyme) to an antigen-specific antibody (Ab) can be used for direct identification of the corresponding antigen. Indirect immunochemical staining utilizes an unlabelled primaiy Ab which is detected by a labelled secondary Ab raised against IgG of the species that produced the primary reagent.

Background labelling, caused by non-specific binding of antibodies to the cell, should be low with direct detection, but staining can be less sensitive than by the indirect method. Cell labelling with conjugated antibodies is quicker; however the coupling of markers to antibodies is time-consuming, expensive, and may result in a loss of activity. The indirect staining technique offers greater versatility of reagents with no loss of activity of the unlabelled primaiy Ab and is more sensitive than direct labelling as the signal is amplified due to the primary Ab having multiple binding sites for the secondaiy Ab. However, there may be higher background staining (see Section 2.2.4), which can be overcome by blocking non-specific binding sites with an appropriate serum. A variation of both protocols is the use of a biotinylated primaiy or secondaiy Ab, which is detected by labelled biotin-binding proteins or anti-biotin antibodies. Titrate primaiy Abs to obtain an optimal specific signal-to-background noise.

2.2.2 Dual and triple immunochemical staining

The simultaneous study of multiple antigens can be achieved by dual and triple labelling. The use of direct immunochemical detection is preferable, however, if not available, indirect methods can be adapted for use.

The primaiy antibodies limit the number of detectable parameters as these should either be differently labelled or raised in a different species. For indirect detection the secondaiy Abs must distinguish between the various primaiy Abs so they should vaiy in species or IgG subclass. A biotinylated form of one of the primaiy Ab may be used (see Section 2.1.1) to facilitate detection if the primaiy Abs are from the same species.

Controls are essential. Single-stained cells must always be included to control for the 'spill over' (bleed-through) for example, of fluorochromes into the range of different fluorescent detectors (see Section 3.1.2).

2.2.3 Permeabilization of cells

If the antigen is predominately expressed on the cell surface, the use of non-permeabilized cells is recommended as there is then less background binding of Ab to intracellular proteins. Permeabilization allows the visualization of intra-

cellular and cell surface antigens. Cells can be permeabilized after fixation (see Section 2.3.3) by 0.1% (v/v) Triton X-100 or 0.25% (w/v) saponin (see Protocol 3). Comparisons between permeabilized and non-permeabilized cells can give an estimate of the distribution of the antigen within a cell.

2.2.4 Secondary antibodies

Macrophages express several receptors that bind the Fc region of both the primaiy and secondaiy Abs. We recommend using Fab2' fragments as secondary Abs to reduce this and to block the cells with normal serum or IgG from the animal used to raise the secondaiy reagent. Labelled secondary Abs are commercially available from Sigma, Serotec, or other commercial suppliers. Table 2 contains a list of fluorochromes commonly attached to proteins or Abs. Secondaiy Abs coupled to alkaline phosphatase, peroxidase, and biotin allow for enzymatic detection of the antigen. These are useful for light microscopic analysis of macrophages in tissues. Endogenous enzyme or biotin can be a problem in different tissues.

Table 2 Fluorochromes and their spectra


Excitation (nm)

Emission (nm)


Fluorescein isothiocyanate (FITC)




Phycoerythrin (PE)




Texas Red (TRSC)



Molecular Probes

Rhodamine (TRITC)




Indodicarbocyanine (Cy5)



Molecular Probes




Molecular Probes

Lucifer yellow




2.2.5 Antibody specificity controls

Indirect immunochemical staining must include a control for the specificity of the primary Ab. Isotype-matched control Abs should be of the same subclass as the primary antibody, but not directed against any cell antigens. Controls for directly-conjugated Abs are more difficult, but an identically labelled non-reactive isotype-matched control should be used. These controls are especially important because of antigen non-specific Fc receptor (CD16, CD32, CD64) binding (see Section 2.2.4). Blocking some Fc receptors with the 2.4G2 antibody (PharMingen) can reduce this background. Where available, excess free antigen, for example a peptide, could be used to show specificity of labelling by a putative antibody.

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