Sometimes antibodies bind to tissue sites that do not contain the specific antigen of interest; this is because the antiserum contains a mixture of antibodies including some from the host animals’ serum. Their detection produces ‘background’ staining. Background staining is an issue, as it may mask staining of the antigen of interest preventing quantification. Background staining can also be caused by cross-reactivity between antibodies raised in varying species. Because of the similarities of immunoglobulin molecules from different species, it is possible that the secondary- layer anti-immunoglobulin will react with the primary-immunoglobulin of the animal in which the antigen is to be immunostained, for example anti-mouse immunoglobulin with rat immunoglobulin (Polak & Noorden, 2005). Most manufacturers test commercially available antibodies for cross-reactivity during development using ELISAs. Product data sheets will then specify any known cross- reactivity and the species which the antibody has been tested against.
There are two main types of controls, positive and negative, which aid in the confirmation of background staining and accuracy of results found.
2.10.3.1 Positive Controls
Positive control samples known to contain the antigens in question can be obtained, often by harvesting appropriate tissue samples at the time of perfuse-fixation. For example, in the studies outlined here, spleen samples were removed during dissection and sections processed and stained alongside brain sections. Spleen was selected as a control tissue for the staining of proliferating cells, as it rapidly regenerates when injured, but also has a high turnover of cells in general (Levy et al., 1976). The pia mater was also used, as an internal brain control, albeit outside the BBB. It is the innermost layer of the meninges, the membrane which surrounds the brain and is
128
lined by epithelial cells, a cell-type which self-regenerates (Decimo et al., 2012). This rapid cell turnover is ideal for demonstrating uptake of BrdU. Without the use of such controls, a negative result on the test material cannot be confirmed as either real or artefact; because there is no guarantee that the reagents are in good working condition and have been applied in the correct order and at the correct dilutions. If the positive control is satisfactory, it is a reasonable assumption that the correct method has been carried out on the test material as well (Heyderman, 1972; Polak & Noorden, 2005).
2.10.3.2 Negative Controls
Negative controls are preparations wherein application of the primary antibody solution is omitted. In all other respects, the treatment is exactly the same as for the test preparation. There should be no staining visible at the end of the reaction. If there is, it must be assumed that it is non-specific, and attempts to prevent it can be made. There are two commonly used solutions to this problem. The first is to dilute the antibody as much as possible prior to use, and the second is to block any potential binding sites with a highly concentrated protein solution. Either of these steps should lessen the effect of background staining mentioned previously. Non-specific binding sites can normally be blocked with normal serum or an ‘inert’ protein such as bovine serum albumin (BSA) or casein, at a concentration of 2% solution in buffer (Polak & Norden, 2005). However, these steps can generally be prevented by suitable wash steps, as these non-specific bonds are weaker than the specific ones (Buffa et al., 1979). Thus, in the studies documented here, working antibody solutions were made from stock solutions containing 0.1% BSA and removed from sections by washing in 10 mM PBS multiple times between incubations (see Section 2.10.4, below).
2.10.3.3 Absorption Controls
To demonstrate that an antibody is binding specifically to the antigen of interest, it can be pre-incubated with the same peptide (immunogen) used to immunise the animals that produced the antibody. This should deactivate the antibody, and the tissue should show little or no staining. This is often referred to as an absorption control or specificity control, and the peptide is often referred to as the blocking peptide (see Figure 2.20). The pre-absorbed antibody can then be incubated with tissue in place of the primary antibody alone. The staining pattern produced by the
129
primary antibody can be compared to that produced by the pre-absorbed antibody (Polak & Norden, 2005).
Figure 2.20. Absorption Control. As in a competitive assay, the immunogen peptide competes with the protein of interest in the tissue sample for antibody binding sites. Source: www.neuromics.com.
Absorption controls work best when the immunogens are peptides. However, if antibodies have been raised against a whole protein, then addition of the mixture of antibody and protein may result in even greater non-specific staining. Therefore, it is important to note that an absorption control using whole protein may not always confirm the specificity of an antibody for the protein of interest in the tissue. An absorption control is essentially a competitive assay, as the peptide competes with the protein of interest for antibody binding sites in the tissue sample (Figure 2.20). Therefore, absorption with excess peptide is preferable, to favour the antibody binding with the peptide while inhibiting binding with the protein of interest.
Both primary and secondary antibodies used in these studies had been well- characterised previously by the manufacturers, ourselves and others (as revealed by consultation with the literature) and were known to stain their target proteins without the need for additional specificity tests.