Staging of Alzheimer disease-associated neurofibrillary pathology using paraffin sections and immunocytochemistry

Assessment of Alzheimer’s disease (AD)-related neurofibrillary pathology requires a procedure that permits a sufficient differentiation between initial, intermediate, and late stages. The gradual deposition of a hyperphosphorylated tau protein within select neuronal types in specific nuclei or areas is central to the disease process. The staging of AD-related neurofibrillary pathology originally described in 1991 was performed on unconventionally thick sections (100 μm) using a modern silver technique and reflected the progress of the disease process based chiefly on the topographic expansion of the lesions. To better meet the demands of routine laboratories this procedure is revised here by adapting tissue selection and processing to the needs of paraffin-embedded sections (5–15 μm) and by introducing a robust immunoreaction (AT8) for hyperphosphorylated tau protein that can be processed on an automated basis. It is anticipated that this revised methodological protocol will enable a more uniform application of the staging procedure.

The AD-related pathological process spans decades and, during this time, the distribution pattern of the lesions develops according to a predictable sequence [8,21,89,93, for a broader discussion of the histopathological diagnosis of AD, see 54,69,76]. A staging system for the intraneuronal lesions introduced in 1991 diVerentiated initial, intermediate, and late phases of the disease process in both non-symptomatic and symptomatic individuals [21, 24-28, 39, 40, 43, 44, 63, 71, 75, 79-82, 84, 85, 87]. In 1997, this staging system was incorporated into the NIH-Reagan criteria for the neuropathological diagnosis of AD [64,66,83]. The Braak system was based upon assessment of two 100 m sections processed according to the silveriodate technique proposed by Gallyas [49-51, 67, 68, 78]. The Wrst section included the hippocampal formation at uncal level, the anterior parahippocampal gyrus, and portions of the adjoining occipito-temporal gyrus. The second section, taken from the occipital neocortex, encompassed portions of the striate area, parastriate area, and peristriate region (Fig. 1). Distinctive diVerences in the topographical distribution pattern of the neuroWbrillary lesions enabled the observer to assign a given autopsy case to one of six stages [21,22]. This simple system had the advantage of being reproducible without having to rely on quantitative assessments or knowledge of patient-related data (age, gender, cognitive status). Furthermore, the result of the silver reactions in unconventionally thick sections provided a means of "reading" a given stage with the unaided eye.
For routine diagnostic purposes, however, such a system is problematic because it calls for unusually thick sections cut from blocks embedded in an unconventional medium [polyethylene glycol (PEG)] [91]. In addition, the method requires that free Xoating sections be stained by experienced laboratory assistants using a non-automated silver technique. These features drastically limit the feasibility of the original staging protocol for routine diagnostic use in the majority of neuropathological laboratories [35]. At the same time, they account for the fact that the staging system has found broad acceptance in a research context while having been subjected to numerous modiWcations [9, 18, 36, 39, 40, 53, 59-62, 69-71, 75, 76, 83, 88]. Neuropathologists in routine diagnostic praxis as well as reference centers that maintain brain banks are interested in a uniform staging procedure so that the material submitted by various participating institutions can be used and evaluated according to the same criteria. Such a staging system must be reproducible, cost-eVective, and easy.
In recent years, sensitive immunocytochemical methods have been developed, the application of which makes it possible to reliably detect not only incipient neuroWbrillary pathology in mildly involved brain regions of non-symptomatic individuals but also, with disease progression, the full extent of the intraneuronal pathology in the end phase [19,90]. NeuroWbrillary changes of the Alzheimer type consist of stable proteins that are impervious to postmortal delay or suboptimal Wxation conditions, and immunoreactions for demonstration of hyperphosphorylated tau protein can be carried out even on tissue that has been stored for decades in formaldehyde [1,72].
Immunoreactions against hyperphosphorylated tau, however, cannot fully replace the Gallyas silver staining method (see Technical addendum), inasmuch as both techniques identify partially diVerent structures. At the beginning of the intraneuronal changes, a soluble and non-argyrophilic material develops, Wlling the somata of involved nerve cells as well as dendritic processes and axons. Thereafter, the distal dendritic segments become snarled and develop dilated appendages. The soluble "pretangle" material is identiWable in immunoreactions for hyperphosphorylated tau protein but remains Gallyas-negative. It is the harbinger of an argyrophilic, insoluble, and non-biodegradable Wbrillary material that emerges after cross-linkage and aggregation of the soluble pretangle material [7,19,96]. The Gallyas-positive neuroWbrillary aggregations gradually Wll the cytoplasm, sometimes inWltrating the proximal dendrites to form a neuroWbrillary tangle, and appear in dendritic segments as NTs without involving the axon [19,45,95]. Following neuronal death, the abnormal material remains visible in the tissue as extraneuronal ghost tangles or tombstone tangles. With time, ghost tangles are no longer immunoreactive for hyperphosphorylated tau protein and their argyrophilia gradually becomes less pronounced [17,19,34]. In summary, the pretangle material can only be detected by immunocytochemistry and this fact has been taken into account in the revised staging procedure presented here (Fig. 2).
In view of the progress that has been made in the demonstration of the neuroWbrillary changes of the Alzheimer type, it seemed expedient to revise the 1991 staging procedure by introducing immunoreactions for visualization of hyperphosphorylated tau and by adapting the tissue selection and processing to the demands of the routine diagnostic laboratory. The goal remains the same, namely to stage the AD-related neuroWbrillary pathology in six stages, as previously, with emphasis this time on the plexuses formed of both pretangle and tangle material, but using paraYn sections immunostained for hyperphosphorylated tau and processed on an automated basis. To illustrate the advantages and disadvantages of both methods, required brain regions with lesions representing AD stages I-VI have been digitally photographed both in silver-and immunostained 100 m PEG sections and in 7 m paraYn sections immunostained for hyperphosphorylated tau protein (AT8-antibody). The revised procedure is intended to facilitate the uniform application of the staging procedure, which now can be performed with greater eYciency than previously.

Fixation and macroscopic preparation
Brains obtained at autopsy should be Wxed by immersion in 10% formalin (4% aqueous solution of HCHO) for one week or longer. Partially remove the meninges to uncover the rhinal sulcus, collateral sulcus, and calcarine Wssure (Fig. 1a-d).
Whereas the original staging procedure requires evaluation of thick silver-stained sections from two relatively large blocks of cortical tissue, the revised version uses immunostained paraYn sections microtomed from three blocks of conventional size that Wt routine tissue cassettes. Figure 1d shows the cutting lines for removal of the three blocks and, in addition, those for the classical view of the hippocampal formation. Alternatively, the tissue on one side of a cut can be used for conventional paraYn embedding (thin sections), and that on the other side for PEG embedding (thick sections).
The Wrst block includes anteromedial portions of the temporal lobe cut at the mid-uncal or amygdala level (frontal section through the temporal lobe at the level of the mamillary bodies) encompassing anterior portions of both the parahippocampal gyrus and adjoining occipito-temporal gyrus. The cutting line runs through the rhinal sulcus (Fig. 1a, d). The sections from this block contain central portions of the entorhinal region and the adjoining transentorhinal region, the latter of which is concealed in the depths of the rhinal sulcus [23,98]. This block is essential for assessment of neuro-Wbrillary AD stages I-III.
The second block simpliWes assessment of stage IV. It is obtained from the same slice as the Wrst block and includes portions of the medial and superior temporal gyri (Fig. 1a, d). As an alternative to the Wrst two blocks, the entire slice through the temporal lobe can be used, provided slides of suYcient size are available. Reduction of this slice to two blocks is recommended to avoid exceeding the size of conventional tissue cassettes.
The third block is removed halfway between the occipital pole and the junction of the parieto-occipital sulcus with the calcarine Wssure. The cut is oriented perpendicular to the calcarine Wssure (Fig. 1c, d). Again, the size of the block has been reduced to Wt standard tissue cassettes. Care has to be taken that the block includes part of the lower bank of the calcarine Wssure and the adjoining basal occipital gyri encompassing portions of the neocortex, i.e., the peristriate region, parastriate Weld, and a clearly deWnable primary Weld, the striate area (Brodmann Weld 17 with the macroscopically identiWable line of Gennari). This block is indispensable for recognition of the neuroWbrillary AD stages V and VI.

Immunocytochemistry
Mounted paraYn sections of 5-15 m thickness are de-waxed and re-hydrated.
The monoclonal antibody AT8 (Innogenetics, Belgium) is one of several commercially available spe-ciWc antibodies that show robust immunoreactivity for hyperphosphorylated tau protein, and a recently published immunocytochemical trial using this antibody has yielded reproducible results [1]. AT8 does not cross-react with normal tau epitopes or require special pre-treatments, and it is exceptionally reliable in human autopsy material regardless of the length of the Wxation time in formaldehyde and/or the condition of the preserved tissue [16,19,57,77]. When performed on paraYn sections (5-15 m), AT8-immunoreactions permit counter-staining for other structures of interest, Fig. 1 Scheme showing the location of the three blocks of tissue required for staging of AD-related neuroWbrillary changes. The Wrst block at the far left (a) includes anteromedial portions of the temporal lobe. It is cut at the mid-uncal or amygdala level (frontal section at the level of the mamillary bodies) and includes the parahippocampal and adjoining occipito-temporal gyri (see enlarged insert below a). The cutting line runs through the rhinal sulcus. The second block comes from the same level and includes part of the medial and superior temporal gyri (a). The third block at the far right (c) is removed from basal portions of the occipital lobe. The cut is oriented perpendicular to the calcarine Wssure. The block includes the neocortex covering the lower bank of the calcarine Wssure and the adjoining basal occipital gyri. It thus shows portions of the peristriate region as well as of the parastriate and striate areas (see enlarged insert below c). provided that diaminobenzidine is used as a chromogen. Homogeneous immunoreactions can also be achieved using PEG sections (50-150 m) (Fig. 2). The sections are incubated for 40 h at 4°C with the AT8 antibody (1:2,000) and thereafter processed for 2 h with the second biotinylated antibody (anti-mouse IgG). Reactions are visualized with the ABC-complex (Vectastain) and 3,3-diaminobenzidine (Sigma).
Prolonged Wxation of brain tissue in a formaldehyde solution may cause metachromatic precipitations (Buscaino bodies or mucocytes) [73]. Components of this material partially react with silver methods and also may interfere with immunoreactions. The precipitations can be removed with pyridine or a tenside solution [1 unit volume Tween 20 (Merck-Schuchardt 822184) and 9 unit volumes de-ionized water] at 80°C for 30 min or both. The sections are then rinsed thoroughly under running tap water and transferred to de-ionized water. Figure 2 is designed to facilitate a direct comparison between selected cortical areas in 100 m thick PEGembedded sections. The Wrst section of each pair has been silverstained according to a modiWed version of the technique originally proposed by Gallyas [22, 31, 49-51, 67, 68, 78], whereas the second serial section (i.e., back-to-back sections from the identical tissue block) underwent staining with the antibody AT8.

Comparison between Gallyas silver-and AT8-immunostained thick (100 m) sections
Intraneuronal neuroWbrillary tangles are visualized with equal clarity by both methods (Fig. 2). Further, the plexuses of argyrophilic NTs are visible not only in the Gallyas sections but also in the AT8-immunostained sections-in the latter, however, the plexuses can be seen to include additional pathologically altered neuronal components (dendrites, axons) that contain non-argyrophilic "pretangle" material (see Fig. 2f, h). Such immunostained plexuses appear much more compact than those depicted by the silver stain, and their obvious advantage is that they can be recognized immediately with the naked eye (see Fig. 3). This applies particularly to the AT8-immunoreactive plexuses located in the deep cortical layers (see Fig. 2v, x, z, zЉ), whereas in Gallyas sections such macroscopic recognition is not always possible (compare Fig. 2v with Fig. 2u).
Nonetheless, the distribution pattern of the immunoreactive cortical alterations throughout the various Welds that are crucial for staging purposes corresponds to that of the argyrophilic lesions ( Fig. 2) and, as such, it allows the observer to trace the progress of the neu-roWbrillary pathology in both silverstained (Fig. 2) and immunostained sections alike (Fig. 3). The greater emphasis on the abnormal plexuses in AT8-immunoreactive sections, however, facilitates the immediate diagnostic assessment of the stages, as, for instance, is readily evident even in the scaled down photographs of the hemisphere sections shown in Fig. 3. These immunopositive plexuses are still visible macroscopically in paraYn sections (5-15 m), and it is helpful, initially, without using the microscope, to view all three slides against a light background to assign them preliminarily to a given stage.
The Wnal diagnosis is essentially based on recognition of the topographical distribution pattern of the neuroWbrillary pathology and calls for a precise knowledge of which regions in the cerebral cortex, in which sequence, develop the AD-related neuroWbrillary lesions. This decision can be made with almost the same degree of accuracy regardless of whether immunostained or silverstained sections are employed, although, based on experience, there is a slight tendency to assign a higher stage to the immunostained slides. The frequency of stage I cases, for example, is somewhat higher in AT8-immunostained sections because in the incipient phases of the disease process AT8-immunopositive nerve cells appear that still lack argyrophilic material. Thus, it is advisable to perform the staging procedure using either the Gallyas or AT8 technique but not both methods. Fig. 2 Comparison of Gallyas silver-and AT8-immunostaining of cortical neuroWbrillary pathology as seen in adjacent serial 100 m polyethylene glycol-embedded sections. The distribution pattern of the lesions throughout the various cortical Welds that are necessary for staging purposes basically corresponds in both methods. It is possible with either technique to assess the progress of the neuroWbrillary pathology. In the revised staging procedure, however, the greater emphasis on the presence of abnormal plexuses, which also include non-argyrophilic pretangle material in AT8-ir sections, facilitates rapid diagnostic assessment of the stages. a-d stage I: Mild involvement is conWned to the transentorhinal region. Note that the plexus of AT8-ir nerve cell processes (b and d) is more conspicuous than that of argyrophilic neuropil threads (a and c). Sections originate from a non-dement- Cases without cortical AD-related neuroWbrillary pathology The transentorhinal region as well as the entorhinal region and hippocampal formation remain devoid of AT8-immunoreactive nerve cells (Fig. 4a, b).

Major characteristics of stages I-II
Stage I: Lesions develop in the transentorhinal region (Figs. 2a-c, 3a, 4c, d) [23] Subcortical nuclei (i.e., locus coeruleus, magnocellular nuclei of the basal forebrain) occasionally show the earliest alterations in the absence of cortical involvement [29]. The transentorhinal region is the Wrst site in the cerebral cortex to become involved. AT8-immunoreactive (ir) projection cells contain hyperphosphorylated tau in both the cell body and all of its neuronal processes (Fig. 4c, d). Late phases of the stage show abundant AT8-ir neurons that permit recognition of the descent of the superWcial entorhinal cellular layer (pre-, i.e., the outer layerof the external principal lamina) from its uppermost position at the entorhinal border to its deepest position at the transition towards the adjoining temporal neocortex (Figs. 1, 3a) [23]. The entorhinal region proper remains uninvolved or minimally involved. (Figs. 2e, f, 3b, 4e) From the transentorhinal region, the lesions encroach upon the entorhinal region, particularly its superWcial cellular layer, pre-(Figs. 1, 3b, 4e-g). The deep layer, pri-, gradually becomes visible (Figs. 2f, 3b, 4e), shows sharply deWned upper and lower boundaries, and is separated from pre-by the broad, wedge-shaped lamina dissecans (myelinated Wber plexus) [23,98]. AT8-ir pyramidal cells appear in sectors 1 and 2 of the hippocampal Ammon's horn (CA1/CA2) (Fig. 3b). Dilations develop transiently in apical dendrites that pass through the stratum lacunosum moleculare of CA1 [20]. Scattered NPs appear in CA1. Fine networks of AT8-ir neurites form in both the stratum radiatum and stratum oriens.

Major characteristics of stages III-IV
Stage III: Lesions extend into the neocortex of the fusiform and lingual gyri (Figs. 2i-n, 3c, d, 4h) The lesions in stage II sites become more severe. The outer entorhinal cellular layers (pre-, pre-, pre-) and most Fig. 3 Stages I-VI of cortical neuroWbrillary pathology in 100 m polyethylene glycol-embedded hemisphere sections immunostained for hyperphosphorylated tau (AT8, Innogenetics). a stage I: Involvement is slight and all but conWned to the transentorhinal region (part of the temporal mesocortex), located on the medial surface of the rhinal sulcus. The section originates from a non-demented 80-year-old female. b stage II: Additional immunoreactivity occurs in layer pre-or layer II of the entorhinal region. The layer gradually sinks into a deeper position in the transentorhinal region (arrow). The border between the entorhinal and transentorhinal regions is clearly recognizable in these early stages (arrowhead). Furthermore, the lesions make headway into the hippocampus (arrow). The section was obtained from a non-demented 80-year-old male. c stage III: The lesions in the hippocampal formation worsen. Entorhinal layers pre-and, additionally, pri-of the deep layers become strongly involved. Lesions extend through the transentorhinal region into the adjoining high order sensory association areas of the temporal neocortex. The lesions generally do not extend beyond the occipito-temporal gyrus laterally (arrow) and lingual gyrus posteriorly. The section originates from a 90-year-old female. d stage III: A Xat section through the entire basal surface of the temporal lobe reveals the severe involvement of the entorhinal cortex (anterior portions of the parahippocampal gyrus) at stage III and shows the tendency of the pathology to extend from there into the adjacent neocortex, i.e., occipito-temporal gyrus laterally (arrow) and lingual gyrus posteriorly (arrow). e stage IV: The third and fourth sectors of the Ammon's horn and a large portion of the insular cortex (arrow) become aVected. The involvement of the neocortical high order sensory association cortex of the temporal lobe now extends up to the medial temporal gyrus and stops short of the superior temporal gyrus (arrow). The primary Welds of the neocortex (see transverse gyrus of Heschl) and, to a large extent, also the premotor and Wrst order sensory association areas of the neocortex remain intact. This section was taken from an 82-year-old demented female. f stage V: In addition to the presence of AD-related lesions in all of the regions involved in stage IV, pathological changes appear in the superior temporal gyrus and even encroach to a mild degree upon the premotor and Wrst order sensory association areas of the neocortex. g stage V: In the occipital lobe, the peristriate region shows varying degrees of aVection, and lesions occasionally can even be seen in the parastriate area. Stage V sections were obtained from a 90-year-old female with dementia. h-i stage VI: Strong immunoreactivity can be detected even in the Wrst order sensory association areas (e.g., the parastriate area) and the primary areas of the neocortex (e.g., the striate area) of the occipital neocortex. Compare the superior temporal gyrus and transverse gyrus of Heschl at stage V with the same structures at stage VI. Both stage VI sections originate from a severely demented 70-year-old female Alzheimer patient. Scale bar applies to all thick sections of the molecular layer become Wlled with intermeshing AT8-ir neurites, whereas the pale lamina dissecans contains a few radially oriented neurites. The deep layer pri-is heavily aVected and gradually thins in the transentorhinal region as it approaches the temporal neocortex (Figs. 3c, 4h). CA1 appears band-like, and transient dendritic changes in CA1 reach their culmination point [20]. CA2 is Wlled with large and strongly AT8-ir pyramidal cells. A moderate number of mossy cells with characteristic dendritic excrescences appear in CA3 and CA4 [97]. The granule cells of the fascia dentata remain uninvolved. AT8-ir sections showing the classical view of the hippocampal formation (Figs. 1b, d) facilitate recognition of lesions in the fascia dentata and Ammon's horn [92].
From the transentorhinal region, the lesions encroach upon the neocortex of the fusiform and lingual gyri, and then diminish markedly beyond this point (Figs. 3d, 4j). An AT8-ir plexus Wlls the cellular layers of the temporal neocortex (Figs. 2n, 4l). The outer line of Baillarger is barely developed and gradually becomes recognizable only with increasing distance from the transentorhinal region. A few NPs develop in the outer layers II-IV.
Stage IV: The disease process progresses more widely into neocortical association areas (Figs. 2o-t, 3e, 5a) Lesional density increases in sites aVected in stage III. A few AT8-ir pyramidal cells appear in the subiculum. The density of the neuritic plexuses of the entorhinal and transentorhinal regions increases and causes a corresponding blurring of the lamina dissecans. The deep plexus spans all of the deep layers: pri-, pri-, and pri-, and from there penetrates widely into the white substance. This aspect of maximum involvement undergoes little change until the end-phase of AD. Thus, the pathological features of the entorhinal and transentorhinal regions must not be taken into account for further diVerentiation of stages V and VI. CA1/CA2 are recognizable as dense bands. The varicose dendritic segments vanish from CA1 without leaving behind any remnants. Large numbers of mossy cells in CA3 and CA4 become AT8-ir. A few AT8-ir granule cells appear in the fascia dentata.
In stage IV, the pathology extends broadly into the mature neocortex. A slight decrease in the immunoreactivity of neocortical NPs can be recognized starting at the border facing the transentorhinal region. Dense neuritic plexuses develop up to the middle temporal convolution (Figs. 2s, t, 4a-c), and a rapid decrease in the severity of the lesions occurs at the transition to the superior temporal gyrus (Fig. 3e). The occipital neocortex is unaVected (Fig. 5e, d) or contains blotch-like local accumulations of AT8-ir pyramidal cells and/or NPs in the peristriate region (Brodmann area 19).

Major characteristics of stages V-VI:
Stage V: The neocortical pathology extends fanlike in frontal, superolateral, and occipital directions, and reaches the peristriate region (Figs. 2u-x, 3f, g, 5f, j) From sites involved at stage IV, the lesions appear in hitherto uninvolved areas and extend widely into the Wrst temporal convolution (Fig. 3f) as well as into high order association areas of the frontal, parietal, and occipital neocortex (peristriate region, Figs. 2v-x, 3g). Initially, unevenly and loosely distributed NPs appear in layers II and III, followed by large numbers of AT8ir pyramidal cells in layers IIIa, b and V. The lower border of the outer neuritic plexus in layers II-IIIab blurs at its transition to the uninvolved layers IIIc and IV (outer line of Baillarger, Fig. 5g). In stage V, the deep plexus of layer V is narrow and tends not to extend into layer VI and the white matter (Fig. 5g, h). The same pattern (only less pronounced) is seen in secondary areas of the neocortex, where uneven accumulations of NPs predominate. AVection of layer V is weak (Fig. 5j). The primary visual Weld (striate area) contains only isolated signs of the pathology consisting of NPs (Fig. 5i, j). Isolated AT8-ir neurons also can be seen in layer IIIab (Fig. 5i, j).
Stage VI: The pathology reaches the secondary and primary neocortical areas and, in the occipital lobe, extends into the striate area (Figs. 2y-zЉ, 3h, i) Most areas of the neocortex show severe aVection and nearly all layers are Wlled with AT8-ir neurites. As such, the outer line Fig. 4 Progress of cortical neuroWbrillary pathology, as seen in paraYn sections immunostained for hyperphosphorylated tau (AT8, Innogenetics). a, b Control case displaying no AT8-ir intraneuronal changes. Note that even the transentorhinal region is devoid of immunoreactivity. c, d stage I: The Wrst AT8-ir pyramidal cells often are more easily detected in thick sections than in paraYn material. Closer inspection of the predilection site (transentorhinal region in d, framed area in c), however, reveals the initial lesions. The meshwork of ir-neurites is as yet not well developed. e-g stage II: Many AT8-ir projection neurons are seen in the transentorhinal region accompanied by a well-developed plexus of ir-neurites (f, g). The pathology also extends into the entorhinal layers pre-and pri-(arrowheads in e). h-l stage III: The transentorhinal and entorhinal regions are more severely involved than in the preceeding stage, and the pathology now extends into the adjoining temporal neocortex of the occipitotemporal and lingual gyri (h, j). The middle temporal gyrus remains uninvolved (i). Scale bar in a, c, e, h is also valid for i and Fig. 5 a, e, f, j, k, o below of Baillarger-a pallid stripe in stage V-begins to blur (Fig. 5l). Layer V still appears as a recognizable band but continues into the neuritic plexus of layer VI. The underlying white substance contains AT8-ir axons. A decrease in immunoreactivity of NPs is seen in many neocortical areas and is most pronounced in the basal temporal Welds. In the occipital lobe, the pathology breaches the parastriate and striate areas (Figs. 2y-zЉ, 3i, 5m-o). Large numbers of NPs and AT8-ir nerve cells appear in layers II and IIIab. Baillarger's outer line or the line of Gennari maintains a light appearance, interrupted only by radially oriented AT8-ir neurites. A sharply drawn AT8-ir plexus follows in layer V (Figs. 2z, zЉ, 5n, o).

Discussion
By applying the silver technique proposed by Bielschowsky [11][12][13][14][15] Alzheimer [2,3] became the Wrst to describe the NFTs that develop in the course of the disease that bears his name. This staining technique has been in use for decades but has been subjected to numerous modiWcations [10,33,36,48,52,74,99]. In systematic studies, Gallyas [49,50] replaced the critical steps of the Bielschowsky technique by means of more manageable reactions and developed a reliable method for selectively demonstrating AD-related neuroWbrillary changes. Following its adaptation for use on 100 m thick sections, this method was standardized for a procedure to stage the development of the cortical AD-related neuroWbrillary pathology that gradually found international acceptance [64,66,83]. Nonetheless, one of the unavoidable pitfalls associated with using silverstaining techniques is that diVerent laboratories produce results of widely varying quality [1]. Subsequently, the 1991 staging protocol, too, underwent a series of permutations, among them the application of various types of silver impregnations, the analysis of cortical sites that are fundamentally less well suited for the procedure, and the use of tissue sections, the thickness of which diVered from that originally proposed [8, 9, 18, 21, 22, 36, 39-41, 53, 54, 59-61, 69-71, 75, 76, 78, 83]. A radical reduction of the diVerent stages also has been suggested [62].
Since AD is an ongoing and not a static process, every staging procedure is, de facto, an artiWcial construct. It is the extent of brain involvement rather than qualitative changes in the neuroWbrillary pathology that increases with disease progression and, as such, the concept of six neuropathological stages (and only six stages) is not entirely amenable to pathological states of a "transitional" nature that do not fulWll the criteria for one of the six neuroWbrillary stages described above.
Here, a revised version of the 1991 staging procedure is presented that can be performed on paraYn sections of conventional thickness, which have been immunostained with the AT8 antibody and processed on an automated basis, thereby fulWlling the demands of the routine laboratory. The simplicity and uniformity of any staging system is the prerequisite for eVective comparisons of results among laboratories and for reliable as well as reproducible classiWcation of a disease process [35,86].

Technical addendum
The previous staging protocol relied upon an advanced but inexpensive silver technique that exploits the physical development of the nucleation sites and in so doing permits careful control of the entire staining procedure [49][50][51]. Insoluble Wbrillary AD-related material can be visualized virtually in the absence of distracting background staining [66,67,78]. The technique can be applied to routinely Wxed autopsy material, even when the material has been stored for decades in formaldehyde solutions. It facilitates processing of large numbers and/or large sections (e.g., hemisphere sections). A homogeneous staining that permeates the entire thickness of a section is achieved even in 50-150 m sections [22]. Thin paraYn sections (5-15 m) can also be used Fig. 5 Progress of cortical neuroWbrillary pathology, as seen in paraYn sections immunostained for hyperphosphorylated tau (AT8, Innogenetics). a-e stage IV: The disease process extends into the high order sensory association neocortex of the temporal lobe (temp.) and includes the medial temporal gyrus (a-c). The peristriate region as well as the parastriate Weld and striate area of the occipital lobe (occ.) still lack the neuroWbrillary pathology (e, d). f-j stage V: Large numbers of neuritic plaques appear in the neocortex (g, h). Pathological changes now encroach to a mild degree upon premotor areas and Wrst order sensory association Welds. In the occipital lobe (j), it is chieXy the peristriate region (h) that shows varying degrees of aVection, and lesions occasionally even develop in the parastriate area. The striate area remains uninvolved (i, j). k-o stage VI: Drastic aVection of the neocortex is seen at stage VI with involvement of almost all areas. Strong immunoreactivity can be recognized even in premotor areas and Wrst order sensory association areas (e.g., the parastriate area m), as well as in primary neocortical areas (e.g., the striate area n). The borderline between the striate and the parastriate areas is drawn easily and-owing to the sudden cessation of the line of Gennari (plexus of myelinated Wbers)-usually can be detected with the unaided eye. A key feature of stage VI is the involvement of the striate area (n), characterized by a dense neuritic mesh in layer V with sharply drawn upper and lower boundaries. Note that the myelin-rich line of Gennari (layer IVb n) is virtually devoid of neuroWbrillary pathology. Scale bar in n also applies to g, h, i, l, m and counter-stained for easy identiWcation of cytoarchitectonic units or speciWc nuclei [31]. NeuroWbrillary changes of the Alzheimer type (NFTs, NTs, NPs) appear in black and, thus, contrast well against an almost unstained background. Connective tissue, glial Wlaments, normal components of the neuronal cytoskeleton, Pick bodies, Lewy bodies/neurites, and corpora amylacea remain unstained. Abnormal tau-protein in argyrophilic grain disease (AGD), in progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), and Niemann Pick type C (NPC) can be visualized as well [46,78].
The staging procedure originally required sectioning at a thickness of 100 m. Silverstained or immunostained sections of such thickness are optimal for the demands of low power (stereo) microscopy and greatly facilitate recognition of the laminar and areal distribution pattern of the lesions (see hemisphere sections in Fig. 3). Sections of this thickness can be gained from non-embedded brain tissue with the aid of a vibratome or a freezing microtome. Alternatively, the tissue blocks can be embedded in PEG [91] and sectioned with a sliding microtome. Application of PEG (400 and 1,000: Merck-Schuchardt 807 485 and 807 488) is rapid, simple, and causes little shrinkage [22].
The blocks are transferred from 96% ethanol to PEG 400 and their surfaces covered with blotting paper. Blocks are placed on a rotating table and after having sunk to the bottom (this can take several days), they are transferred to fresh PEG 400 for an additional day. Then, transfer to PEG 1000 at 54°C for 1 day. Embed in fresh PEG 1000, mount, and section at 50-150 m. Transfer sections to 70% ethanol to remove the embedding medium. Store sections in formaldehyde solutions. Prior to staining, transfer sections to de-ionized water. It is important to note that the Gallyas silver technique displays only highly aggregated Wbrillary material, whereas the AT8immunoreaction also visualizes the non-argyrophilic material that initially develops within involved neurons (pretangle material).