ARTICLE |
Correspondence to: Wing Gee Liu, Inst. for Animal Health, Neuropathogenesis Unit, Ogston Building, West Mains Road, Edinburgh UK EH9 3JF, Scotland. E-mail: winggee.liu@bbsrc.ac.uk
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Summary |
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One of the pathological changes characteristic of the transmissible spongiform encephalopathies (TSEs) is the accumulation of disease-specific PrP (PrPsc). Immunolabeling of PrPsc was compared using a panel of monoclonal and polyclonal antibodies. To determine the effects of tissue fixation on immunostaining, we performed a supplementary investigation reviewing the fixatives formol saline and periodatelysineparaformaldehyde (PLP). The main target sites of the antibodies were similar. However the monoclonal antibodies (MAbs) 6H4, 7A12 and 8H4 revealed targeted PrPsc labeling with no background labeling. Although 7A12 and 8H4 did not detect early PrP deposition, we propose that during the later stages of disease 7A12 and 8H4 can be used with equal effectiveness in place of 6H4. Tissues taken during the early stages of disease that had been fixed in PLP displayed more PrP immunolabeling than tissues that had undergone formol fixation. PLP fixation on 6H4-immunostained tissue revealed interweaving granular linear PrP deposits in the hippocampus. This labeling was not observed in tissue that had undergone formol fixation, suggesting that PLP fixation might enhance the sensitivity of the immunohistochemical (IHC) detection of PrP. In the two scrapie mouse models studied here, PLP fixation and immunolabeling with the anti-PrP antibody 6H4 gave superior results.
(J Histochem Cytochem 51:10651071, 2003)
Key Words: scrapie, CNS, PrP, immunohistochemistry
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Introduction |
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TRANSMISSIBLE SPONGIFORM ENCEPHALOPATHIES (TSEs) are a group of fatal neurodegenerative disorders that include bovine spongiform encephalopathy (BSE) in cattle, CreutzfeldtJakob disease in humans, and scrapie in sheep and goats. Naturally occurring in sheep and goats, scrapie demonstrates the pathological changes typical of the TSEs such as vacuolar damage, neuron loss, astrogliosis, and abnormal deposition of the disease-specific form of the normal cellular prion protein (PrPc). The conversion of PrPc into the abnormal protease-resistant scrapie prion protein (PrPsc), a cell-surface sialoglycoprotein with a concentrated ß-sheet confirmation, and its accumulation in the brain are neuropathological characteristics of these diseases. The immunohistochemical (IHC) detection of these accumulations provides a reliable clinical diagnostic marker for the TSE diseases (
To further clarify the role of PrP during disease pathogenesis, the deposition of disease-specific PrP has been studied in experimental murine scrapie models using IHC techniques. In normal mice, BSE-infected mice, and mice infected with scrapie, PrP is associated with the follicular dendritic cells of the spleen, lymph nodes, Peyers patches, and the islets of Langerhans in the pancreas (
Immunolabeling of PrP using the antibodies 1A8 and 1B3 (
Recent investigations have also used the mouse MAb 6H4 (Prionics; Zurich, Switzerland) to reveal PrP deposition in the CA2 region of the hippocampus in the 87V/VM model of scrapie (
The two scrapie mouse models used here differ in their targeting of PrPsc deposition in the brain. The ME7 scrapie strain produces widespread diffuse vacuolation and PrP accumulation of varying intensities throughout the brain (
The principal aim of our investigation was to determine if there were variations in PrPsc immunolabeling using five PrP antibodies and if any differences were evident at various stages of disease. We compared the immunolabeling of anti-PrP monoclonal and polyclonal antibodies in the ME7/CV experimental murine scrapie model over several time points. We also compared the immunolabeling of anti-PrP monoclonal and polyclonal antibodies in the 87V/VM murine scrapie model at the 80-day point to reproduce the early PrP labeling observed in previous investigations (
It has been previously reported that PLP-fixed brains of mice can reveal extensive PrPsc immunoreactivity in comparison to brains fixed in formol saline (
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Materials and Methods |
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The ME7 murine scrapie model was produced by intracerebrally injecting the F1 cross between the C57BL/DK and VM/DK mouse strains (known as CV) with 20 µl of a 1% (w/v) brain homogenate from a C57BL mouse terminally infected with the ME7 strain of scrapie. This model develops consistently severe hippocampal pathology (
The 87V murine scrapie model was produced by intracerebrally inoculating VM/Dk mice with 20 µl of a 1% (w/v) brain homogenate from a mouse terminally infected with the mouse passaged 87V scrapie strain. The 87V scrapie-infected and normal brain-injected control mice were sacrificed by cervical dislocation and their brains removed at 80 dpi and terminal point of disease (320 dpi).
Once removed, the brains were fixed in 10% formol saline or PLP and trimmed to give four coronal levels [approximating Figures 25, 46, 64, and 85 in
The pretreatments applied are based on the standard immunolabeling protocols for 1A8, 1B3 and 6H4 used at the Neuropathogenesis Unit (NPU) to optimize labeling and minimize nonspecific background staining during immunolabeling in mouse brain, and has been referred to in previous publications (
With combined pretreatments of formic acid immersion (
To reduce the risk of unwanted background labeling, endogenous peroxidase activity was inhibited with hydrogen peroxidase in methanol. For the polyclonal antibodies, nonspecific binding sites were blocked by preincubation in normal goat serum at 1:20 (Diagnostics Scotland; Edinburgh, UK). For the MAbs, the nonspecific binding sites were blocked by preincubation in normal rabbit serum at 1:20 (Diagnostics Scotland).
Primary antibodies 1A8, 1B3 (NPU) and 6H4 (Prionics) were applied optimally titrated and diluted to 1:2000; 7A12 and 8H4 (kindly provided by Dr. Man-Sun Sy of Case Western Reserve University; Cleveland, Ohio) were diluted to 1:800. The sections were exposed to the primary antibodies overnight at room temperature. For the polyclonal antibodies, negative control sections were incubated with normal rabbit serum (Diagnostics Scotland). The secondary antibody was biotinylated goat anti-rabbit (Jackson ImmunoResearch, Stratech Science; Luton, UK) used at 1:400 for 30 min. For the MAbs, negative control sections were incubated with normal mouse serum (Diagnostics Scotland). The secondary antibody was biotinylated rabbit anti-mouse (Jackson ImmunoResearch) used at 1:400 for 30 min. All sections were incubated in avidinbiotin complex (ABC) Elite kit PK-6100 (Vector Laboratories; Peterborough, UK) and the reaction product was visualized with 0.025% diaminobenzidine (Sigma; Dorset, UK).
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Results |
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The five anti-PrP antibodies were compared at different stages of disease and the results are summarized in Table 1 and Table 2. Differences between the antibodies were more noticeable during the earlier stages of disease. In the ME7/CV scrapie model at 70 dpi, 1A8 (Fig 1A), 1B3 (Fig 1B), 6H4 (Fig 1C), 7A12 (Fig 1D), and 8H4 (Fig 1E) detected the deposition of PrP in the pyramidal cell body layer of the CA1 region of the hippocampus. However, the intensity of staining of the sections stained with 1A8 (Fig 1A) and 1B3 (Fig 1B) was compromised in the neuropil by the nonspecific binding partly concealing PrP labeling.
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During the progression of disease, visualization of the increasingly widespread diffuse PrP pathology in the ME7 model was minimally affected by nonspecific labeling. This was initially noticeable at 96 dpi in the hippocampus of sections stained with 1A8 (Fig 1F), 1B3 (Fig 1G), 6H4 (Fig 1H), 7A12 (Fig 1I), and 8H4 (Fig 1J). Although the background labeling masked the early diffuse PrP staining in the CA1 region of the hippocampus (Fig 1AE), the deposition of PrP by 96 dpi was sufficiently established to counteract this effect.
In the 87V/VM murine scrapie model which has targeted PrP pathology, during the terminal stages of disease 1A8 (Fig 1K) and 1B3 (Fig 1L) labeled sections showed nonspecific background staining that partially masked the PrPsc labeling. However, 6H4 (Fig 1M), 7A12 (Fig 1N), and 8H4 (Fig 1O) revealed intense and specific PrPsc labeling of the amyloid plaques, fine granular depositions, and diffuse accumulations, with no evident nonspecific background staining. During the early stages of disease (80 dpi), 6H4 (Fig 2A), 1B3 (Fig 2B), and 1A8 (not shown) all detected early punctate PrP deposition in the dorsal raphe, median raphe nucleus, and vestibular nucleus, with no apparent difference in targeting or intensity. In contrast, 7A12 and 8H4 did not detect this early PrP deposition (not shown).
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PLP-fixed 87V/VM murine scrapie brains demonstrated early PrP staining at 80 dpi in the vestibular nucleus, dorsal raphe, and median raphe nucleus with 6H4 (Fig 2A), 1B3 (Fig 2B), and 1A8 (not shown). However, PrP deposition was not detected with 7A12 or 8H4 (not shown). PrPsc immunolabeling was not revealed at 80 dpi in 87V/VM brains fixed in formol saline with any of the antibodies tested (Fig 2C). This fixative effect is not repeated with the ME7/CV murine scrapie model. Sections stained with 6H4, 1B3 and 1A8 showed no such difference at 51 dpi (polyclonal results not shown), where PrP was observed in the dorsal raphe of brains fixed in PLP (Fig 2D) and formol saline (Fig 2E). As in the early stages of disease in the 87V/VM scrapie mouse model, the early deposition of PrP in the ME7/CV model was not detected by 7A12 and 8H4 when tissues were fixed in either formol saline or PLP (not shown).
We also observed that only PLP-fixed tissues immunostained with 6H4, 1B3, and 1A8 revealed delicate tracking of PrP along processes in the stratum radiatum of the hippocampus (Fig 2F) (polyclonal results not shown).
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Discussion |
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Few studies have concentrated on determining if there are variations in PrPsc immunolabeling using different PrP antibodies at various stages of disease. We show that the main targeting sites of 1A8, 1B3, 6H4, 7A12, and 8H4 in the brains of ME7 scrapie-infected mice were similar, with PrPsc labeling observed in the same neuroanatomic areas in the later stages of disease. Similarly, in 87V scrapie-infected mice, the main target sites of the five antibodies studied were identical in the later stages of disease.
However, differences emerged during the early stages of disease in both murine scrapie models studied. Only 1A8, 1B3, and 6H4 detected early punctate PrP deposition in the 87V scrapie model at 80 dpi and the ME7 scrapie model at 51 dpi. These results are consistent with reports that 87V scrapie-associated PrP is first detected at 70 dpi (
That the monoclonal antibodies 7A12 and 8H4 did not label early PrP deposition is not an indication of their lack of efficacy but does lend support to the complex nature of PrP expression as various glycoforms in different brain regions (
Previous reports that PLP-fixed brains of mice reveal extensive PrPsc immunoreactivity have been partially supported by this investigation. Tissues that had been fixed in PLP displayed more PrP immunolabeling than tissues that had undergone formol fixation. Although during the early stages of disease in the ME7/CV model there is no significant difference in PrP immunolabeling with fixation in either PLP or formol saline, a subtle difference between the effects of the two fixatives on immunolabeling was apparent on closer inspection.
A delicate tracking of PrP was seen in the stratum radiatum of the hippocampus, similar in appearance to the interweaving granular linear PrP deposits observed and described in more detail by
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Conclusion |
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The principal aim of our investigation was to determine if there are differences in PrPsc immunolabeling using different PrP antibodies. We report that there is no discernible difference between the targeting of the monoclonal antibody 6H4 and the polyclonal antibodies 1A8 and 1B3 in the scrapie-infected mouse brain. Furthermore, we report that the MAbs 7A12 and 8H4 do not detect the early deposition of PrP. However, it is conceivable that the targeted nature of 7A12 and 8H4 is directed towards specific PrP glycoforms that may not be expressed during the onset of disease and early PrP deposition.
Comparing the five anti-PrP antibodies studied, we suggest that 6H4 is a more appropriate antiserum for PrP immunodetection in the mouse CNS throughout the course of disease, revealing targeted PrPsc labeling with no nonspecific reaction. Given the panel of antibodies currently available for PrP immunodetection, we also propose that 7A12 and 8H4 can be used with equal effectiveness in place of 6H4 for PrP immunolabeling in the later stages of disease in the mouse CNS. That 6H4 targets a single epitope in the PrP sequence and demonstrates comparable targeting to 1B3 and 1A8 that targets multiple epitopes suggests that monovalency does not result in weaker immunodetection. It appears that it is the location of the epitope on the PrP molecule to which the antibody is targeted that ultimately determines the accuracy of immunodetection.
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Acknowledgments |
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We thank Patricia McBride (Neuropathogenesis Unit) for helpful discussion. We also thank Drs Man-Sun Sy and Rona Barron for kindly providing the antibodies 7A12 and 8H4.
Received for publication November 25, 2002; accepted April 16, 2003.
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