ARTICLE |
Correspondence to: Guoying Bing, 310 Davis Mills Building, Dept. of Anatomy & Neurobiology, University of Kentucky Medical Center, Lexington, KY 40536-0098. E-mail: gbing@pop.uky.edu
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Summary |
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An improved thioflavin-S stain, Gallyas silver stain, and two immunostainings were quantitatively compared for demonstration of neurofibrillary tangles (NFTs) on the same sections. Sections of hippocampal formation from seven cases of Alzheimer's disease (AD) were immunofluorescently stained with a commercially available polyclonal NFT antibody or a PHF-1 monoclonal antibody, followed by an improved thioflavin-S stain, and finally by Gallyas silver staining. The thioflavin-S method was improved by using a combination quenching method that removes background autofluorescence without remarkable tissue damage and by post-treatment with concentrated phosphate buffer, which minimizes photobleaching. PHF-1 or NFT immunostaining is much less sensitive than the improved thioflavin-S staining and Gallyas silver staining, particularly in the transentorhinal region. Moreover PHF-1 immunoreactivity varied greatly among AD individuals. Thioflavin-S staining and Gallyas silver staining show almost the same sensitivity in NFT demonstration, but only the former depends on the secondary protein structure of NFTs. This study suggests that the improved thioflavin-S staining is a simple, sensitive, and consistent method for demonstration of neurofibrillary pathology. (J Histochem Cytochem 50:463472, 2002)
Key Words: Alzheimer's disease, neurofibrillary tangles, transentorhinal cortex, thioflavin S, silver staining, immunohistochemistry
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Introduction |
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Neurofibrillary tangles (NFTs) are a major defining neuropathological feature of Alzheimer's disease (AD) and other tangle-bearing disorders. Because postmortem diagnosis of AD relies in part on the accurate detection of NFTs in the brain, a number of methods have been developed to visualize these neurofibrillary lesions in brain sections. NFTs consist of bundles of paired helical filaments (PHFs) and straight filaments (
Comparisons of the various staining methods for NFT demonstration had been reported in several earlier studies (
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Materials and Methods |
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Tissues
Postmortem brain tissues were obtained from 7 AD patients autopsied at the University of Kentucky Alzheimer's Disease Research Center (ADRC). Age at death of AD patients was 79.2 ± 8.7 years (range 6591) and that of controls was 79.9 ± 9.1 years (range 6695). The mean postmortem intervals were 2.8 hr (range 2.73 hr) for AD brains and 2.9 hr (range 1.84.5 hr) for controls. All AD subjects met the clinical and neuropathological criteria for the diagnosis of AD. Control subjects were individuals without evidence of neurological disorders who had been followed longitudinally as part of the normal volunteer control group at the University of Kentucky ADRC. Paraffin-embedded blocks were prepared by sequential dehydration in graded ethanol and vacuum-infiltration in paraffin before embedding and serial sectioning to a thickness of 8 µm.
Antigen Retrieval and Quenching Autofluorescence
Deparaffinized brain sections were rehydrated in distilled water and placed in a stainless steel pressure cooker containing a boiling dilution (1:100) of Antigen Unmasking Solution (#H-3300; Vector Laboratories, Burlingame, CA) according to the manufacturer's protocol. After heating under pressure for 1 min, samples were removed, placed briefly in a room-temperature (RT) water bath, and washed in PBS pH 7.2, for 5 min.
Because the human brain sections showed considerable autofluorescence at FITC and Cy3 channels because of the abundance of highly fluorescent lipofuscin granules, a combination quenching procedure was used to reduce background autofluorescence (
Immunofluorescent Staining
After antigen retrieval and quenching treatment, the deparaffinized sections were subjected to conventional immunofluorescence staining using either rabbit anti-NFT polyclonal antibody (#AB1518; Chemicon, Temecula, CA) or mouse PHF-1 monoclonal antibody, which recognizes the dually phosphorylated Ser396 and Ser404 epitope of tau peptides (
Improved Thioflavin-S Staining
Traditional thioflavin-S staining was previously modified by
In our same-section comparison study of multiple staining methods, the thioflavin-S staining was preceded by the combination quenching steps (described above) and conventional immunofluorescent staining on deparaffinized sections. Sections were then stained with 0.05% thioflavin-S in 50% ethanol in the dark for 8 min. In preliminary optimization trials, dilutions of 0.01%, 0.05%, 0.1%, and 1% thioflavin-S solutions were tested for optimal staining quality at 8 min. A thioflavin-S concentration of 0.05% was found to be optimal and was used for the remainder of the study. This step was followed by differentiation in two changes of 80% ethanol for 10 sec each and three washes in large volumes of distilled water. Slides were then incubated in a high concentration of phosphate buffer (411 mM NaCl, 8.1 mM KCl, 30 mM Na2HPO4, 5.2 mM KH2PO4, pH 7.2) at 4C for 30 min or more, then briefly rinsed with distilled water and coverslipped using water. No mounting resin was used so as to facilitate re-use of the same sections for subsequent stains. Images at different subregions were captured with a digital camera, and NFTs were counted (see below) before any subsequent silver staining procedure.
Fluorescence Microscopy
The induced fluorescence, silver staining, and immunostaining were viewed using an Axioplan 2 imaging microscope (Zeiss; Oberkochen, Germany). For fluorescence, the filters used were as follows: rhodamine (ex 546 ± 6 nm, em > 570 nm); FITC (ex 470 ± 20 nm, em >515 nm); DAPI (ex 365 ± 6 nm, em >397 nm). Images were captured using an Axiocam digital camera interfaced with a computer containing Axiovision 3.0 software (Zeiss). Inputs from rhodamine and FITC channels were superimposed to facilitate comparison.
Gallyas Silver Staining
Gallyas silver staining (
NFT Density
Typical fields containing high tangle densities were chosen for comparison at three representative regions (CA1, subiculum/presubiculum, and transentorhinal cortex). The numbers of NFTs were counted within the frame of captured images (1340 x 1060 µm2) at x200 magnification. Histological cues within a section were used to facilitate selection of identical fields so that the counting frame was set at the same positions for the comparison of the three methods. NFT density was calculated as the number of NFTs in the frame divided by the area of the frame. In the transentorhinal region, we followed the pattern of lamination described by
Formic Acid Treatment
Formic acid destroys the secondary structure of proteins immediately without cleaving or removing the protein (
Statistics
NFT densities are expressed as mean ± SE. ANOVA was used to test the significance of difference in NFT densities detected by the three methods.
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Results |
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Improvement of the Thioflavin-S Staining Method
We report a substantial improvement of the thioflavin-S technique that eliminates background cellular autofluorescence, minimizes photobleaching effects, and reduces tissue damage associated with NaOH and acetic acid pretreatment in deparaffinized sections. In the conventional thioflavin-S method, as modified by
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Comparison of Immunostaining, Thioflavin-S Staining, and Gallyas Silver Staining on the Same Sections
In this study we compared NFT immunostaining, thioflavin-S staining, and Gallyas silver staining in paraffin-embedded sections of various regions of the AD brain. Comparisons were made on the same sections to avoid possible misinterpretations caused by differences among sections and to permit detection of more subtle differences in specificity or sensitivity. Sequential application of anti-NFT immunohistochemistry, our improved thioflavin-S staining, and the Gallyas silver staining revealed co-detection of some tangles in hippocampal CA1 (Fig 2A12A4, arrows) and subiculum/presubiculum (Fig 2B12B4, arrows). However, the polyclonal anti-NFT antibody rarely detected NFTs in the pri- layer of transentorhinal cortex (Fig 2C12C4), where most NFTs appear early in AD (
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The monoclonal PHF-1 antibody, which recognizes tau protein phosphorylated at serine residues 396 and 404, detected NFTs in both the hippocampus and transentorhinal cortex (Fig 3). However, greater variations in PHF-1 immunoreactivity were observed among AD individuals. Some AD cases showed very few PHF-1-positive NFTs but had a considerable number of NFTs detectable by thioflavin-S. This variation was also reflected in the quantitative comparisons as larger standard errors for PHF-1 staining compared with those for staining with polyclonal NFT antibody (see Table 1). Even in cases where abundant NFTs were stained with PHF-1 antibody (Fig 3), PHF-1 immunostaining still detected fewer NFTs than thioflavin-S.
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Staining with Gallyas silver and thioflavin-S on the same fields revealed that these two methods detected the same NFTs (Fig 2C2D). Some NFTs displayed only very faint staining in 2/14 sections examined (Fig 4B, arrowhead) and in their adjacent sections stained with Gallyas silver alone (data not shown). Gallyas silver staining appeared to detect more neuropil threads (Fig 4A and Fig 4B) but only weakly stained amyloid plaques. In addition, our preliminary results using a recently developed intrinsic fluorescence induction method (
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Quantification of NFTs showed that the thioflavin-S and Gallyas silver stains identified the same number of NFTs in the three regions examined. In the CA1 and subiculum regions, polyclonal NFT antibody or PHF-1 antibody recognized approximately half of the NFTs detected by the other two methods (Table 1). In the transentorhinal/entorhinal cortex, PHF-1 immunostaining detected more NFTs than the polyclonal NFT antibody (Table 1), although it still left out many NFTs that could be detected by thioflavin-S staining and Gallyas silver staining.
Dependence of Staining on Secondary Structure
Because the thioflavin-S and Gallyas silver staining showed comparable sensitivity in demonstrating NFTs (Table 1), these two methods might share a similarity in staining mechanisms. To address this question, we investigated whether ß-pleated sheet secondary structure was necessary for both methods. When applied after treatment with formic acid, which disrupts the structure of ß-pleated sheets without cleaving or removing the protein (
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Discussion |
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In this study we further improved the thioflavin-S staining method by introducing several technical modifications that result in overall reduction of tissue damage, photobleaching, and background autofluorescence, with no loss of sensitivity or selectivity. Although the modification of the thioflavin-S technique by
The results of this study show that NFT or PHF-1 immunostaining is much less sensitive than thioflavin-S or Gallyas silver staining in detecting NFTs. The polyclonal anti-NFT antibody used in the present study was created by immunizing against a set of NFTs that were extracted in boiling SDS and purified by sucrose gradient centrifugation (
It should also be noted that the PHF-1 antibody used in the present study detects a specific phosphorylated epitope at serine residues 396 and 404, near the C-terminus, of the tau protein. Although the pathways and key phosphorylation events leading to tau hyperphosphorylation in vivo are poorly understood, in vitro studies show that hyperphosphorylation of tau is part of the mechanism of self-assembly into tangles of PHFs and straight filaments (
Our study showed clear region-specific differences in NFT and PHF-1 immunoreactivity, with transentorhinal and entorhinal cortical areas yielding the fewest immunopositive tangles. Based on the known neuroanatomic distribution of NFTs as a function of AD progression (
The greater sensitivity of the thioflavin-S and Gallyas silver staining methods compared to anti-tau immunohistochemistry warrants further inquiry into the mechanisms of these stains. The related benzothiazole dye thioflavin-T (ThT), a classical amyloid stain for senile plaques containing ß-amyloid peptide in AD brain, binds rapidly and specifically to anti-parallel ß-sheet fibrils. The fibrillar ß-sheet-bound dye species undergoes a characteristic 120-nm red shift of its excitation spectrum that may be selectively excited at 450 nm, resulting in a fluorescence signal at 482 nm (
The mechanisms of Gallyas silver staining of NFTs have not been fully elucidated. It does not bear an absolute requirement for ß-sheet structures because it can stain NFTs in formic acid-pretreated sections. One possibility is that Gallyas silver staining may be related to the loss of the ability of NFTs to reduce silver ions in a manner similar to that of degenerative axons (
In terms of applicability to routine neuropathological diagnosis, the general affinities of thioflavin-S for diverse AD pathologies can be viewed as a technical advantage because it can stain both plaque and tangle pathology with a single and simple technique. Usually, difficulty in long-term preservation of thioflavin-S results limits its wide application in routine neuropathological diagnosis of AD, although the ability to digitally store photomicrographs may obviate this drawback. Our modifications to the procedure, which enhance tissue quality and fluorescence photostability, may make the method more consistent and therefore more amenable to routine clinical use. The Gallyas silver stain is another sensitive method for NFT demonstration but, like other silver stains, is often associated with higher variability than thioflavin-S. One drawback is that, unlike fluorescence-based methods, it lacks the capacity for multiple labeling. Our results with immunostaining suggest that tau immunoreactivity using the two antibodies examined may not be useful for quantitative staining of tangles, although they can be useful in differentiating specific NFT subpopulations. Finally, our general approach to performing multiple stains on same sections may be useful in a number of research situations where individual tangles may need to be identified or categorized based on their affinities for certain antibodies.
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Acknowledgments |
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Supported by NIH grant 3R01 NS39345-01 (to GB).
Human brain sections used in this study were kindly provided by the University of Kentucky Alzheimer's Disease Research Center, headed by Dr William R. Markesbery. Ann Tudor cut the sections for this study.
Received for publication July 11, 2001; accepted November 28, 2001.
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