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A New Specific, Sensitive And Non-carcinogenic Reagent For The Demonstration Of Horseradish Peroxidase

J. Hanker, P. Yates, C. Metz, A. Rustioni
Published 2005 · Chemistry, Medicine

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3,3'-Diaminobenzidine (DAB), introduced by Graham & Karnovsky (J. Histochem. Cytochem. 1966, 14, 291 & J. exp. Med. 1966, 124, 1123) is one of the most widely employed reagents in histochemistry. It has been very useful in demonstrating the sites to which the exogenous ultrastructural tracer horseradish peroxidase (HRP) is transported in vertebrate tissues. Over the past five years, DAB staining of transported HRP in neurons has developed into a powerful neuroanatomical tool for labelling the cells of origin of pathways in the central nervous system (La Vail, 1975, in: The use o f axonal transport for studies o f neuronal connectivity (eds. W. M. Cowan & M. Cuenod), pp. 217-248. Amsterdam: Elsevier.). Reports of the borderline carcinogenicity of DAB in rats (Griswold, Casey, Weisburger & Weisburger, Cancer Res. 1968, 28,924) may be responsible for the decreased availability of good quality DAB. Recent studies (Hanker, Anderson & Bloom, Science 1972, 175, 991; Hanker & Rabin, J. clin. Mierobiol. 1975, 2, 463) suggested that oxidative coupling reactions of aromatic amines in the presence of phenols might provide a suitable substitute for DAB. Some of these reactions yield deeply-coloured synthetic melanin-like compounds which are osmiophilic and sufficiently insoluble to be suitable end-products for histochemistry. In addition, the reaction must be sufficiently rapid to deposit the end-product at the sites of the plant hydroperoxidase alone. Such a reaction was realized when it was found that the peroxidation of p-phenylenediamine (PPD) was greatly accelerated by the presence of pyrocatechol (PC). The co-polymer formed as a result of this oxidative coupling reaction was osmiophilic and was bluer than oxidized DAB. It was insoluble and conformed well to biological ultrastructure. Neither PPD nor PC used individually gave satisfactory results and the best results were obtained when a mixture of 1 part (by weight) PPD to 2 parts PC was employed as the reagent. The administration of HRP and fixation of tissue for studies in mice, other than those involving axonal transport, were carried out according to Graham & Karnovsky (J. Histochem. Cytochem. 1966, 14, 291 &J. exp. Med. 1966, 124, 1123). For the demonstration of retrograde axonal transport in cats, rats, and rhesus monkeys, 30% HRP (Boehringer) in distilled water was injected into the sensorimotor cortex, or the ventrobasal complex of the thalamus (VB injection) or in the cerebellar cortex. The volume of HRP solution injected varied from 0.05 to 2/al. The animals were perfused with a mixture of 0.5% (para)formaldehyde and 2.5% glutaraldehyde in 0. t M phosphate buffer, pH 7.2. Tissue blocks (brain) were excised and immersed in 30% sucrose in 0.1 M phosphate buffer (pH 7.2) immediately after the perfusion. Cryostat or Vibratome* sections of mouse tissues were cut at 10-20/~m. Frozen sections of the rat, cat or monkey brains were cut at 40/2m.

This paper is referenced by
Sensitivity of the short-range spinal interneurons of the cat to experimental spinal cord trauma.
G. Benoist (1979)
Comparison of alternative chromogens for renal immunohistochemistry.
K. Sheibani (1981)
Pubertal changes in the medio-basal hypothalamic area after neonatal suprachiasmatic nucleus lesions in the rat
E. Marani (2005)
Cellular and spatial changes in the anuran superior olive across metamorphosis
T. Templin (2005)
Corticospinal tract collaterals to the dorsal column nuclei of cats
A. Rustioni (2004)
Griffonia simplicifolia lectins bind specifically to endothelial cells and some epithelial cells in mouse tissues
L. Laitinen (2005)
Immunological analysis of nuclear lamina proteins
R. Stick (2004)
Insulin or insulin-like peptides in the human pituitary.
G. Budd (1987)
The innervation of the rabbit's anterior eye segment: a retrograde tracing study.
M. P. T. ten Tusscher (1988)
Fiber connections of the torus longitudinalis in a teleost: Cyprinus carpio re‐examined
H. Ito (2003)
Sensitivity and specificity of immunohistological methods on freeze-dried paraffin sections.
Z. Nemes (1982)
Quantitative morphological changes in phrenic and intercostal motor columns and their respective spinal cord segments during postnatal development in the kitten
D. Rose (1983)
A comparison of the structures of α‐ and γ‐spinal motoneurones of the cat
D. Westbury (1982)
methods. Sensitivity in horseradish peroxidase neurohistochemistry: a comparative and quantitative study of nine
Marsel M. Mesulam (2013)
Experimental analysis of the transdifferentiation of visceral to parietal endoderm in the mouse
Y. Ninomiya (2005)
Histochemical diagnosis of Hirschsprung's disease and a comparison of the histochemical and biochemical activity of acetylcholinesterase in rectal mucosal biopsies.
W. J. Patrick (1980)
Visuotectal projections following temporary transplantation of embryonic eyes to the body in Xenopus laevis.
N. Munro (1982)
J. P. C. Dumont (1987)
Axonal pathfinding in the absence of normal pathways and impulse activity
W. Harris (1984)
Gene Transfer into Rat Brain Using Adenoviral Vectors
C. Thomas (2001)
44-kDal bone phosphoprotein (osteopontin) antigenicity at ectopic sites in newborn rats: kidney and nervous tissues
M. Mark (2004)
The development of facial motoneurones in the mouse--neuronal death and the innervation of the facial muscles.
K. Ashwell (1983)
Neuronal death during development in the isthmo‐optic nucleus of the chick: Sustaining role of afferents from the tectum
P. G. Clarke (1985)
Topographical distribution of Purkinje cells in the uvula and the nodulus projecting to the vestibular nuclei in cats
H. Shojaku (1987)
Plasticity of non‐giant flexion circuitry in chronically cut abdominal nerve cords of the crayfish, Procambarus clarkii.
M. Lee (1984)
Minimally differentiated acute myeloid leukemia (AML-M0): comparison of 25 cases with other French-American-British subtypes.
A. Venditti (1997)
Nucleotide and DNA uptake by Neurospora crassa: involvement of an uptake stimulating protein.
G. Tóth (1991)
Electrophysiological and morphological properties of rat abducens motoneurones
J. Durand (2004)
Integration in descending motor pathways controlling the forelimb in the cat
B. Alstermark (2006)
Identification of the teleost Edinger-Westphal nucleus by retrograde horseradish peroxidase labeling and by electrophysiological criteria
J. C. Wathey (2004)
Histochemical localization of horseradish peroxidase in neurones
Y. Olsson (1978)
Ganglion cell distribution in the retina of the mouse.
U. Dräger (1981)
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