Cellular localization of p65 immunostaining in brain
Of the tested p65 antibodies, the polyclonal Santa Cruz sc-372 antibody showed the strongest, most repeatable pattern in western blots. In most blots, a single band appeared in the location marked by the 70-kDa lane marker. The single band was completely absent in the RelA knockout (p65 KO) tissue, in brain, liver, and spleen samples. A similar pattern was found with the NIH p65 antibody. The four antibodies that met this rigorous test of specificity were all raised against a portion of the C-terminus of p65, which includes its transactivation domain (TAD). In contrast, antibodies raised against the N-terminus or against the NLS region did not meet the criteria for specificity. Thus, it is apparent that the C-terminus contains antigenic peptide sequences that are unique to p65, whereas other regions of the molecule are not so selectively and strongly antigenic. The NLS region should be notably difficult to raise selective antibodies against because the aa sequences are conserved for a number of proteins that have NLSs. The N-terminal Rel homology domain (RHD) is conserved across other proteins, so this too may not be a suitable region for antibodies. Finally, not surprisingly, the rabbit monoclonal antibodies gave excellent results. They are new and have not been used in many publications to date.
The sc-372 antibody has been used in several publications to characterize NF-κB activity in the brain. A study by [
15] examined the effects of intracerebroventricularly injected KA-induced NF-κB activity in the hippocampus. This study is one of few to show a western blot. The illustrated molecular weight range was confined to 51-80 kDa, and in that range was a single band. Intensity of the band increased in tissues from animals surviving from 1 to 7 days. Immunohistochemistry of the CA3 region of the hippocampus revealed induction of staining in glial cells with the appearance of astrocytes or microglia (co-labeling with both cell types was confirmed by double immunostaining with GFAP and CD11b, respectively), and the staining was found in both the nucleus and cytoplasm. This pattern of staining was closely mimicked by the staining with the NIH p65 antibody (Figure
6c-e). It also agrees with the astrocyte-like cellular staining in hippocampus 4 days after KA injury in another study using the sc-109 antibody [
16], which we find to authentically mark p65 but also additional proteins in the western blot.
In human brain infarctions, a selective staining of glial cells was seen by Terai [
17] using the sc-372 antibody. In the infarct area, microglial cells and blood vessels were also stained. The entire cytoplasm of the cell appeared stained, and such staining was largely absent outside the infarct area.
In a study of status epilepticus seizures, which are associated with neuronal loss, p65 staining with sc-372 was induced in astrocytes and blood vessels mainly in the hippocampus and entorhinal cortex [
18]. Constitutive staining was minimal. There was no overlap of p65 staining and fluorojade staining of degenerating neurons.
Following immune challenge by i.p. IL-1β injection, Nadjar et al. used the sc-372 antibody for IHC in rats and mice to examine the cellular CNS response [
19]. Background staining was low, and induction of staining was seen in non-neuronal cells of the choroid plexus, circumventricular organs, and blood vessels. Co-localization with astrocytes was positive. The temporal course of activation, peaking in the 1-2 hour range, was mirrored also by IκBα mRNA analyzed by RT-PCR. The localization, cell type, and timing of this response was rather perfectly matched by studies of IκBα mRNA induction visualized by ISHH after peripheral injections of lipopolysaccharide [
20] or IL-1β [
21]. IκBα is one of the family of inhibitory proteins that hold NF-κB in the cytoplasm until it is activated [
1]. Activation results in degradation of IκBα, which is followed typically by increased transcription and synthesis of IκBα to replace the reserve of inhibitory molecules. Thus, IκBα mRNA induction is a reliable marker of NF-κB activity in cells [
20]. Significantly, no published ISHH study has shown neuronal induction of IκBα mRNA in any model of brain injury or other form of CNS activation.
Other IHC publications are more difficult to interpret. Quite a few studies do not provide sufficient information in the Method section to allow determination of which specific antibody was used. A study by Gabriel [
22] does not specify which Santa Cruz rabbit polyclonal p65 antibody was used (sc-109 or sc-372), but a western blot showed a single band, suggesting it was sc-372. Dilution of primary antibody was 1:1,000. In the study, control cortex showed staining of the cell cytoplasm in neurons, astrocytes and microglia. After an infarct, endothelium and glial cells were strongly stained in the ischemic area. Nuclear staining was predominant in astrocytes, microglia, and macrophages. In a different study of cortical injury using the sc-109 antibody for IHC, combinations of neuronal and non-neuronal cells were positively stained in the region of injury [
23]. Similarly with this antibody, a diffuse neuronal staining pattern was replaced by a more specific staining of astrocytes and microglia in the region of a cortical injury induced by local injection of NMDA [
24,
25]. A study of ischemia in the gerbil showed exclusively glial (astrocytes and microglia) p65 staining in the hippocampus with the monoclonal antibody (sc-8008) from Santa Cruz [
26]. This antibody showed specificity in cultured neurons in the KO test (figure
2a). Results must be interpreted with caution in work using p65 antibodies that detected a band at the correct molecular weight in WT but not KO tissues, but also marked bands at other mobilities; these other proteins might be stained in IHC material.
In summary, studies employing the antibody we have characterized here as the best one for localizing authentic p65 in brain have fairly consistently shown that p65 staining is not appreciably present in neurons and is not induced in neurons after various brain insults. Rather, p65 immunostaining is increased in endothelia, astrocytes, and microglia in these studies.
In contrast to the above summary, another group of studies shows predominantly neuronal p65 staining, both constitutively and after pathological insults. The most widely used antibody for IHC to depict this pattern of NF-κB activity in the brain is the MAB3026 antibody against "activated" p65. The original p65 antibody raised against the NLS region was a rabbit polyclonal made by P. A. Baeuerle [
27] and named α-p65NLS. Later, an affinity purified monoclonal antibody of the IgG3 isotype, called α-p65M [
28] and α-p65Mab [
29], was used by the Kaltschmidts in early publications and then distributed (as clone 12H11) by Boehringer-Mannheim (1697 838), Roche (1697838), and finally licensed by Chemicon (Millipore) and sold as MAB3026. It was made against the human p65 sequence CDTDDRHRIEE
KRKRT (NLS underlined). The authenticity of the monoclonal antibody was determined by western blot [
10]. Interestingly, in normal 293 cells, a single band appeared at around 67 kDa, but in 293 cells with elevated p65 levels due to transfection with a CMV-p65 plasmid construct, a dense band appeared at 67 kDa and a lighter band appeared just below it, at around 65 kDa. Similar multiple banding patterns were seen in western blots in a subsequent publication [
30]. In light of the present western blot data showing that the band marked by MAB3026 runs just above the band marked by sc-372, it is possible that the faint lower band is the authentic p65, and the major upper band is an unknown protein with biological properties very similar to p65.
In the early years, the MAB3026 antibody was used to support the argument that there was constitutive and induced NF-κB activity in neurons [
28,
31]. Very high concentrations of the antibody were typically used--in the 1:30 to 1:150 dilution range. With this antibody, injury-induced neuronal staining was shown in cerebral cortex and the hippocampal CA fields at 1-3 days after middle cerebral artery occlusion [
32] or 1-2 days after traumatic brain injury (TBI) [
33] and in the hippocampal CA fields 1 day after preconditioning by transient ischemia or KA administration [
34]. In human brain, the MAB3026 antibody strongly stained cortical neurons in the ischemic area following a stroke [
35]. These data are very different than the published data in similar models described above that used the sc-372, sc-109, and sc-8008 antibodies.
Following peripheral lipopolysaccharide (LPS) administration eliciting an inflammatory response in the brain, ISHH showed strongly induced IκBα mRNA expression in non-neuronal cells of the meninges, blood vessels, brain parenchyma, and circumventricular organs [
20,
21]. In striking contrast, in this same LPS administration paradigm, IHC of p65 staining using MAB3026 showed exclusively neuronal induction in one brain region adjacent to one circumventricular organ [
36]. It is not known what factors would produce this pattern of staining.
Neuronal immunostaining with p65 and p50 antibodies
We show here that even antibodies that pass the test of specificity on western blots still label cells nonspecifically when used for IHC. The probable reason for this is that the levels of p50 and p65 in neurons are so low that they cannot be detected by this method. Raising the concentration of the primary antibody to the point that cellular staining becomes visible amounts to nonspecific staining, which we see even at 1:500 dilution of authenticated antibodies. Antibodies that mark additional proteins in the western blots are even more difficult to interpret when used for IHC, especially in reports of changes in staining levels induced by various manipulations. It is unlikely that the changes measured are due to alterations in levels of p65 or p50. Any of the other proteins recognized by these antibodies might contribute to the immunohistochemical outcome. We (Listwak et al., unpublished data) and others [
43] have found that NF-κB in neurons is rather unresponsive to stimulatory challenges of all kinds. Consequently, recent reports of altered NF-κB activity inferred from changes in subunit immunostaining following mild challenges like electrical stimulation of afferent pathways [
44], passive avoidance learning [
45], or psychological stress [
46] need to be viewed with great skepticism. These finding are further rendered suspect by the use of the MAB3026 antibody [
44,
45,
47], the use of unidentified Santa Cruz p65, p50, and IκBα antibodies without designation of catalog numbers [
46], and by the fact that the brain focus of the studies is the hippocampus, which has layers of very tightly packed neurons that render the appearance of specific staining, especially at low magnification, because of the high neuronal packing density. With antibodies such as sc-372 and NIH p65, we find that neurons are the least stained and most unresponsive of all the cellular phenotypes in the hippocampus but are visible because of the high packing density (note CA3 cell layer in Figure
6b,
6d).
The close functional association of p50 with p65 should result in similar immunohistochemical staining patterns in tissues following manipulations. A few studies have used p50 antibodies other than sc-114 for IHC in brain. Some used antibodies distributed by Warner Greene (Gladstone Institute). A p50 antibody named Ab 392 raised against the N-terminus was used in several IHC publications following its characterization in COS cells [
48]. A western blot of brain extracts showed a band at 50 kDa plus multiple additional bands [
49]. Dying hippocampal neurons were immunostained in that study and in a similar study that also used an N-terminus-directed p65 antibody from Greene called Ab 567 [
50]. In another study by the same group, after cerebral infarct, both p50 and p65 antibodies from Greene stained neurons of all types in the infarct area with little staining outside the infarct area [
51]. Neuronal staining with the p50 Ab 392 antibody appeared in one study in which every neuron of the medial septum was immunostained in the normal young brain, and the staining did not change following deafferentation by fornix transection [
52]. It is difficult to assess the significance of these studies without knowing whether or not the antibodies were really specific for p50. Further work with authenticated p50 monoclonal antibodies is warranted.
A p50 antibody made by Mariagrazia Grilli (CNR Institute of Neuroscience, Milan, Italy) but not commercially available passed the test of marking single bands at 50 and 105 kDa in a western blot that were absent in p50 KO tissue, and it was used to show staining of radial glial cells in the subventricular zone and in the rostral migratory stream [
53]. The authors did not show absence of such immunostaining in p50 KO tissue, however.