NR ANWI
AU Kovacs,G.G.; Flicker,H.; Budka,H.
TI Immunostaining for ubiquitin: efficient pretreatment
QU Neuropathology and Applied Neurobiology 2003 Apr; 29(2): 174-7
PT Letter
VT
Immunostaining for ubiquitin: efficient pretreatment
The first description of the presence of ubiquitin, a small stress-induced protein, which participates in the degradation of short-lived and damaged proteins [5], in diverse filamentous inclusions of neurodegenerative disorders has opened a new era in research [11]. The concept of conformational diseases [1] further highlighted its role, thus recently even the term 'ubiquitin protein catabolic disorders' was suggested to designate these abnormalities [8]. Immunohistochemistry (IHC) for ubiquitin (for a review see Kovacs and László [7]) is a well-established screening method in the diagnosis of neurodegenerative diseases, especially in view of the fact that some of this immunoreactivity (IR) for ubiquitin is the major diagnostic lead for the neuropathologist [9]. The most widely used antibodies have a high affinity for ubiquitin protein conjugates and are much less sensitive in detecting free ubiquitin. The more and more widespread international, multicentric brain-banking possibilities require harmonization of methodology and consensus of diagnoses. However, no particular recommendation on IHC for ubiquitin has been reported.
When attempting to identify the most reliable pretreatment for ubiquitin IHC, we investigated different neurodegenerative diseases using four distinct antigen retrieval methods. We included three brains each of CERAD [13] definitive Alzheimer's disease (AD), diffuse Lewy-body disease (DLBD) [12], Parkinson's disease (PD), motor neurone disease (MND) and sporadic Creutzfeldt-Jakob disease (sCJD) with complete neuropathological investigation. We examined the hippocampus and temporal cortex in AD and DLBD, the cingulate cortex in DLBD, the mesencephalon in PD, the spinal cord in MND, and the temporal cortex and cerebellum in sCJD. We applied four different pretreatments: (i) 10 min and (ii) 20 min antigen retrieval using a microwave oven at power 250 W and citrate buffered saline (pH 6.0), (iii) pretreatment (i) followed by 5 min 96% formic acid, (iv) 5 min 96% formic acid, as well as no pretreatment, to adjacent sections. In addition to the antiubiquitin (polyclonal, 1 : 200, Dako, Denmark) antibody, we used anti-tau (monoclonal AT8 1 : 200, Innogenetics, Belgium), anti-alpha-synuclein (monoclonal, 1 : 20, Alexis Biochemicals, San Diego, USA) and anti-prion protein (PrP) (monoclonal 3F4, 1 : 300, Senetek, Maryland Heights, USA) antibodies. As the secondary system we used the ChemMate(TM) DAB detection kit (DAKO, Denmark).
All pretreatments were suitable to reach a neuropathological diagnosis. However, the best pretreatment proved to be protocol (ii). In addition to stronger and wellcontrasted IR, this revealed more small Lewy-body like inclusions in cortical neurones, granular dot-like IR in the neuropil and neurites corresponding to alpha-synuclein pathology in DLBD (Figure 1 a-c), and more neuropil threads and dystrophic neurites in AD (Figure 1 d-f) as compared to sections without pretreatment (Figures 1 and 2 b,e,f). Furthermore, in sCJD cases we noted significant difference in the immunolabelling of kuru plaques (Figure 1 g-i), coarse synaptic (Figure 2 a-c) and patchy/perivacuolar (Figure 2 d-f) type of PrP IR [2,6,14]. In MND spinal cords more dot-like IR was observed surrounding neurones (Figure 2 g,h). In addition to dot-like IR in the white matter, the occasional dispersed cytoplasmic and nuclear, as well as the condensed type of nuclear stainings of cells [7], were also better distinguishable and appeared to be more (Figure 2i,j). In relation to nuclear ubiquitin IR, which is still a matter of debate, earlier observations suggested that it is seen mainly in fresh and not in formalin-fixed, paraffin-embedded material [10] while it was readily visualized with our protocol in paraffin sections. We are currently investigating the role of this type of IR in diverse conditions. There was a marked increase of the detectability of smaller, dense and ubiquitinated-specific (e.g. tau/neuropil threads, alpha-synuclein/Lewy neurites) structures, and in fact the overall signal-to-noise ratio was much improved. Thus protocol (ii) not only gives more confidence in the diagnostic procedure, but also provides new insight into research on the role of ubiquitin in cell death pathways, protein aggregation or antigen presentation. The need for more detailed evaluation of ubiquitin IR is well exemplified, for example, by a recent study on multiple sclerosis [3].
In summary, we suggest that 20 min antigen retrieval in citrate buffered saline significantly increases the reliability of ubiquitin IHC. Application of such optimized protocols might help to improve inter-rater agreement in the neuropathological evaluation of central nervous system disorders [4].
G. G. Kovacs
H. Flicker
H. Budka
Institute of Neurology, University of Vienna and Austrian Reference Centre for Human Prion Diseases, Vienna, Austria
References
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Received 13 March 2002
Accepted after revision 5 August 2002
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Figure 1. Comparison of ubiquitin immunohistochemistry using 20 min antigen retrieval in citrate buffered saline (
a,d,g) to no pretreatment (b,e,h) in diffuse Lewy-body (DLBD) (a-c), Alzheimer's (AD) (d-f), and sporadic Creutzfeldt-Jakob (sCJD) (g-i). Disease-specific pathology is immunolabelled by alpha-synuclein in DLBD (c), by tau in AD (f) and by prion protein in sCJD (i). Original magnification: x10 (a-f), x20 (g-i).
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Figure 2. Comparison of ubiquitin immunohistochemistry using 20 min antigen retrieval in citrate buffered saline (
a,d,g,i) to no pretreatment (b,e,h,j) in sporadic Creutzfeldt-Jakob (sCJD) (a-f) and motor neuron disease (MND) (g-j). Disease-specific pathology is immunolabelled by prion protein in sCJD (c,f). Original magnification: x20 (a-h),x40 (i,j).
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AD Institute of Neurology, University of Vienna and Austrian Reference Centre for Human Prion Diseases, Vienna, Austria
SP englisch