Appendix. Effect of tissue fixation on the quality of immunohistochemistry: comparison of three antibodies in three differently fixed tissues
To illustrate the principles discussed in this review, tissue from four adult male mice (three C57Bl6/J mice; RCC, Füllinsdorf, Switzerland and one GABAA receptor α3 subunit-knockout (GABRA3-null) mouse;Yee et al., 2005) was prepared after perfusion–fixation under deep pentobarbital anaesthesia (50 mL/kg, i.p.). All animal experiments were approved by the local authorities (Cantonal Veterinary Office of Zurich). Except for slight variations of the perfusion and postfixation, procedures were identical for all mice. In particular, the same fixative solution [4% paraformaldehyde in 0.15 m sodium phosphate buffer, pH 7.4, containing 15% of a saturated solution of picric acid (final concentration 0.2%); Fritschy & Mohler, 1995) was used for perfusion.
For mouse 1 (wild-type) and mouse 2 (GABRA3-null), the perfusion cannula was inserted through the left ventricle into the ascending aorta; a constant flow (10 mL/min) of phosphate-buffered saline (10 mL) followed by ice-cold fixative (50 mL) was established with a peristaltic pump, resulting in a rapid fixation monitored by the increase in body rigidity. The brain was extracted immediately after the perfusion; it had a firm consistence and was yellow due to the picric acid in the fixative. It was postfixed in the same solution for 4 h at 4°C.
Mouse 3 (wild-type) was perfusion-fixed exactly like mice 1 and 2, but postfixation of the extracted brain lasted for 18 h.
In mouse 4 (wild-type), the cannula was inserted into the left ventricle only and the perfusion flow was slow and irregular (3 mL/min), resulting in a weak fixation, as judged by the rigidity of the tail and body musculature. The extracted brain was pale and soft, confirming the weak fixation. It was postfixed for 4 h, like for mice 1 and 2.
Tissue cryoprotection, sectioning and the immunoperoxidase staining procedure were performed identically for the four mice, as described (Fritschy & Mohler, 1995), using the Vectastain Elite kit (Vector Laboratories, Burlingame, CA). Primary antibodies included a guinea pig antiserum against the γ-aminobutyric acid (GABA)A receptor α3 subunit (see Studer et al., 2006 for characterization), a mouse monoclonal antibody against cholecystokinin (CCK; CURE/UCLA Digestive Disease Center University of California, Los Angeles, USA; Ohning et al., 1994) and a rabbit polyclonal antibody against serotonin (coupled to bovine serum albumin with paraformaldehyde; Immunostar, Stillwater, MN, USA; product Nr. 20080). These antibodies were selected because they are, in our experience, differentially sensitive to fixation. For controlling the specificity of the α3 subunit antiserum, mouse 2 lacked the GABAAα3 subunit (a GABRA3-null mutant, see Yee et al., 2005). Antibodies were diluted in Tris-saline buffer (pH 7.4) containing 2% normal goat serum to block non-specific binding sites and 0.2% Triton X-100. Sections were processed free-floating, mounted on glass slides, coverslipped and photographed with a digital camera. For details of the staining procedure, please refer to Fritschy & Mohler (1995) and Fritschy et al. (1998).
Figure 1A–D illustrates the dependence of GABAA receptor α3 subunit-immunoreactivity on fixation and the presence of antigen in transverse sections through the amygdala. Specific staining was observed in mice 1 and 4, with the highest signal-to-noise ratio being obtained in the weakly fixed tissue (mouse 4; Fig. 1D). In contrast, variable cytoplasmic staining was evident in the section from the GABRA3-null mouse (Fig. 1B), indicating that this antiserum binds non-specifically to cytoplasmic constituents in the absence of its target antigen. A similar pattern was evident in mouse 3, notably in the basolateral nucleus (Fig. 1C), suggesting that prolonged postfixation causes a dramatic loss of immunoreactivity in the neuropil and appearance of non-specific staining of neuronal somata.
Figure 1. Differences in immunohistochemical staining pattern for the GABAA receptor α3 subunit resulting from tissue fixation, as illustrated in transverse sections through the amygdala processed for immunoperoxidase staining. Sections shown in (A), (B) and (D) were postfixed for 4 h. The boxed panel (D) indicates the highest signal-to-noise ratio, obtained after weak fixation. The specificity of the α3 subunit-immunoreactivity shown in (A), (C) and (D) was verified by staining a section from a GABRA3-null mouse (α3 subunit-ko; B). Note the loss of diffuse staining in the neuropil and the appearance of strong cytoplasmic staining caused by prolonged postfixation (C). Abbreviations: Bl, basal nucleus of the amygdala; Ce, central nucleus of the amygdala; CPu, caudate-putamen; Den, dorsal endopiriform nucleus; La, lateral nucleus. Scale bar: 250 μm.
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Comparing Fig. 1B and C might lead to the premature conclusion that the α3 subunit antiserum is not suitable for immunohistochemistry, because it produces mainly background staining. Novel, or unknown, antibodies should be tested routinely with various protocols to determine their sensitivity to fixation. Finally, note that the background staining seen in the GABRA3-null mouse can be completely eliminated by antigen-retrieval in citrate buffer (Fritschy et al., 1998; Yee et al., 2005).
Figure 2A–C illustrates immunoperoxidase staining for serotonin in the CA1 area of the hippocampus. It is best preserved in rapidly perfused tissue after short postfixation (mouse 1), revealing a homogeneous plexus of fine varicose axons in the stratum oriens and radiatum, being more dense in stratum lacunosum-moleculare (Fig. 2A). Prolonged postfixation (mouse 3) resulted in a different morphology and regional distribution of immunopositive axons, which appeared coarser and considerably less dense in the stratum oriens than in stratum radiatum (Fig. 2B). These alterations were accompanied by increased background staining, notably in the pyramidal cell layer. Finally, immunodetection of serotonin was precluded in the weakly fixed tissue (mouse 4), in which the background was even more intense, notably in the stratum lacunosum-moleculare (Fig. 2C). In this case, the strong background likely reflects vesicular release of serotonin during the weak perfusion–fixation procedure, emphasizing the need of a fast fixation to avoid such artefacts.
Figure 2. Differences in immunohistochemical staining pattern for serotonin (5-HT; A–C) and cholecystokinin (CCK; D–F) resulting from tissue fixation, as illustrated in transverse sections through the CA1 area of the hippocampus processed for immunoperoxidase staining. In each series, the boxed panel indicates the best staining pattern. The morphology and density of serotonin-immunoreactive axons are altered after prolonged fixation (B), and they are undetectable in weakly fixed tissue (C). The arrowheads in (A) point to coarse beaded axons. In contrast, CCK-immunoreactivity in cell bodies of interneurons (arrowhead) and axons innervating CA1 pyramidal cells are detected only after prolonged postfixation (E). Abbreviations: slm, stratum lacunosum moleculare; so, stratum oriens; sp, pyramidal cell layer; sr, stratum radiatum. Scale bars: 100 μm (A–C); 50 μm (D–F).
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Figure 2D–F illustrates the innervation of CA1 pyramidal cell somata by CCK-positive axon terminals, known to belong to a specific population of basket cells (Freund & Katona, 2007). Most strikingly, these axons are detectable by immunoperoxidase staining only following a prolonged postfixation (mouse 3; Fig. 2E), whereas in the regularly fixed tissue (mouse 1; Fig. 2D) no staining is evident, and in the weakly fixed tissue (mouse 4) strong background staining occurs in the stratum oriens and stratum radiatum (Fig. 2F). The dependence of this monoclonal antibody on strongly fixed tissue for proper CCK immunostaining suggests that fixation might influence the conformation of its epitope. Because the perfusion used here is relatively brief, it is possible that CCK might be detectable using a different fixative or perfusion protocol. In any case, immunohistochemical detection of CCK in axon terminals of the hippocampus is delicate and we failed to obtain positive results with a commercially available polyclonal antibody.
These three examples underscore the fact that an accurate description of the distribution of an antigen in brain tissue sections might require comparison of various fixation and tissue-processing protocols. Previous knowledge of function and distribution of a molecule of interest provides crucial help for evaluating its distribution pattern and establishing whether these procedures should be improved. With regard to the three markers investigated here, the following points were considered.
The perfusion procedure can strongly impact the strength of fixation. All mice were properly perfused, as evidenced by the complete absence of erythrocytes (which express endogenous peroxidase activity and become strongly labelled in immunoperoxidase experiments). The weak fixation of mouse 4, deduced from the soft consistency of the tissue, could have been achieved also with a fixative containing a lower concentration of paraformaldehyde.
As a membrane protein participating to synaptic protein complexes, the GABAA
receptor α3 subunit should not be detected predominantly in cell somata (as in Fig. 1C
), whereas diffuse staining of the neuropil reflects its dendritic distribution. White matter is expected to remain totally unstained.
Serotonin in the hippocampus is only present in axons, as serotonergic cells are located in the raphe nuclei; anterograde tracing studies have shown that fine varicose axons originate from the dorsal raphe nucleus and coarse axons from the median raphe nucleus (Freund et al., 1990
). Both types of fibres are evident only in Fig. 2A
CCK is a neuropeptide expressed by a subset of GABAergic interneurons in the hippocampal formation, notably by a subpopulation of basket cells. Failure to detect it (as in Fig. 2D and F
) most likely represents false-negative results, as confirmed by Fig. 2E
A direct consequence of these observations is that multiple immunofluorescence staining experiments require in principle antibodies that have similar requirements for tissue fixation and processing. According to our results, it would be problematic, for instance, to combine staining for CCK and the GABAA receptor α3 subunit in the same section (or even in the same animal). Development of alternative fixation and tissue preparation procedures might be necessary to overcome such constraints. For example, we have shown recently that several limitations due to tissue fixation can be alleviated in tissue sections derived from living brain slices (Schneider Gasser et al., 2006).
Finally, these examples show that the effect of tissue fixation and processing on the apparent distribution of antigens in immunohistochemistry can be a major limiting factor for quantitative analyses, even for relative comparisons. The tissue sections illustrated in Fig. 2A and C are from mice that were perfusion-fixed with the same solution and processed under identical conditions, yet yielding dramatically different results. The only difference between these mice was the perfusion procedure (placement of the cannula and flow rate of the fixative). Slight variations in perfusion–fixation between animals are almost unavoidable in practice. The serotonin staining illustrates that such variations can dramatically affect the apparent density of serotonergic axons in the forebrain.