Brains and Brawn - Antibodies in Research and Diagnosis

07. September 2009

The increased sensitivity of immunohistochemistry (IHC) over standard Hematoxylin and Eosin (H&E) staining has established the IHC technique in the study of many diseases. Herein we describe the utility of some NovocastraTM antibodies that can be used in the study of brain and muscle diseases.

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Dementia is a common, chronic and progressive condition of several different types. It is a complex spectrum of disease states with no single cause. Dementia is characterised by the long-term decline in memory, neuropsychiatric and behavioural disorders. It is common in the elderly, but can occur at any age. Dementia affects 5-8 % of all people ­between ages 65 and 74, and rises with increasing age to 25 %. More than 24.3 million people are estimated to have dementia globally, with 4.6 million new cases being diagnosed each year.

At the molecular level, dementias have been recently classified according to the presence of abnormal proteins such as tau and synuclein.

Fig. 1: Human brain, Alzheimer’s disease: immunohistochemical staining for tau protein using NCL-TAU-2 (Clone Tau-2). Paraffin section.

Alzheimer’s disease (AD) is the most common age-related cause of neuro-degenerative disease and is defined by the presence of the protein tau. The progressive death of nerve cells in the brain is associated with twisted strands (neurofibrillary tangles) of the tau protein (Fig. 1) and deposits of the protein fragment beta-amyloid in amyloid plaques (Fig. 2).

Parkinson’s disease is a progressive, neuro-degenerative condition characterised by the loss of dopaminergic neurons in the midbrain substantia nigra. The abnormal presence of the protein alpha-synuclein has been demonstrated in Parkinson’s disease (PD) and dementia with Lewy bodies (DLB). Lewy bodies are protein aggregates of alpha-synuclein together with others such as ubiquitin, neurofiliament protein and alpha-B crystallin (Fig. 3).

Fig. 2 Human brain, Alzheimer’s disease: immunohistochemical staining for beta amyloid protein using NCL-B-Amyloid (Clone 6F/3D). Note intense staining of senile plaques. Paraffin section.
Fig. 3: Human brain, Lewy body dementia: immunohistochemical staining for alpha-synuclein using NCL-ASYN (Clone KM51). Note staining of alpha-synuclein containing Lewy bodies. Paraffin section.

There is considerable overlap between these disease states where concomitant DLB and AD pathology is present, the so-called Lewy body variant of Alzheimer’s disease. Other proteins, such as amyloid precursor protein (APP) (Fig. 4) and tyrosine hydroxylase (TH) (Fig. 5), may also be useful in the detection of pathogenesis in Alzheimer’s disease.

Fig. 4: Human brain, Alzheimer’s disease: immunohistochemical staining for amyloid precursor protein using NCL-APP-288 (Clone 3G12). Note intense staining of neurofibrillary tangles and senile plaques. Paraffin section.
Fig. 5: Human midbrain: immunohistochemical staining of tyrosine hydroxylase enzyme using NCL-TH (Clone 1B5). Note cytoplasmic staining of catecholaminergic cells and their processes. Paraffin section (Peroxidase substrate: nickel DAB, Counterstain: eosin).

Muscular dystrophy

Muscular dystrophy is characterised by the progressive weakness and degeneration of skeletal muscles. Some patients may only experience mild symptoms within a lifetime whereas others will have severe symptoms and die at a young age. The ­muscular dystrophies share common muscle ­histology demonstrating a characteristic variation in muscle fibre size, fibre death, infiltration by inflammatory cells and ultimately, replacement of the muscle ­fibres by fat and connective tissue. In combination with routine microscopic examination of a muscle biopsy, immunohistochemistry can be used to make a definitive diagnosis in some muscular dystrophies by identifying deficiencies of key proteins.

Of the nine types of muscular dystrophy, the most common and severe form is Duchenne muscular dystrophy (DMD) which affects young boys. It occurs in around 1 in 3,500 male births and is inherited on the X chromosome. Although females may be carriers of the defective gene, they usually show no symptoms. In severe DMD, the dystrophin protein that stabilizes the muscle membrane during contraction is absent resulting in impaired function of the muscle cells and deterioration of the muscle fibres (Fig 6).

Fig. 6: Human skeletal muscle: immunohistochemical staining for dystrophin using NCL-DYSA (Clone 13H6). Note membrane staining of normal muscle fibers (A) and reduced variable staining of muscle fibres from an individual with Duchenne and Becker muscular dystrophy (B). Paraffin section.

In Becker muscular dystrophy (BMD), the second most common form of muscular dystrophy, the dystrophin molecule is also affected but patients show a less pronounced reduction in protein expression. BMD is also linked to the X chromosome, occurring in around 1 in 30,000 male births.

The other types of muscular dystrophy are rare with proteins such as emerin, sarcoglycans and calpain affected and not all are linked to the X chromosome. A differential diagnosis may be achieved by labelling muscle biopsies with antibodies to these proteins.


Nigel Piggott, Ph.D.

Nigel Piggott, Senior Principal Scientist, Leica Microsystems, Newcastle, acquired his Ph.D. at the Edinburgh University in 1979. His doctoral thesis with the title ‘Synthesis of bacterial alginate by Pseudomonas aeruginosa’, sponsored by Tate & Lyle, was honored with the CASE Award.  After working as a researcher for the University of Newcastle upon Tyne, he joined the former Vision Biosystems in 1994 as a Senior Research Associate. In 2002, Nigel Piggott became Antibody Development Manager, before becoming Principal Development Scientist in 2007. He has been holding his present position as Senior Principal Scientist since 2008.

Gary McIntosh, Ph.D.

Gary McIntosh, Technical Project Specialist, Leica Microystems Newcastle, was born in Hull, UK. He graduated with a B.Sc. in Biomedical Sciences from the University of Bradford in 1992. He then studied for a Ph.D. at the University of Newcastle upon Tyne with a thesis on “Evaluating the Significance of Gene Product Expression of an Oncogene Amplification Locus in Human Breast Cancer”. In 1996, he joined Novocastra Laboratories as a monoclonal antibody development scientist. Following the acquisition by Vision Biosystems in 2002, he became the antibody research and development manager. More recently, he is operating as a part of the Leica Biosystems R&D management team to provide technical expertise to maximise the sites efficiency in managing technically complex projects.