Stabilization of tetramethylbenzidine (TMB) reaction product at the electron microscopic level by ammonium molybdate

Marfurt, Carl F.; Turner, Dennis F.; Adams, Catherine E.

doi:10.1016/0165-0270(88)90136-7

Cited by 32 publications

(12 citation statements)

References 16 publications

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“…Unfortunately, such sections are not very useful for examining m o r p h ological details. A n alternative is to use T M B to visualize the H R P , because the H R P / T M B reaction product is clearly visible as an electron-dense crystalline reaction p r o d u c t (Marfurt et al 1988).…”

Section: Discussionmentioning

confidence: 99%

“…Tissue sections were cut from blocks of visual cortex using a vibratome. These sections were then reacted with either diaminobenzidinc (DAB) (Graham and Karnovsky 1966) or with tetramethylbenzidine (TMB) (Marfurt et al 1988). The DAB reaction product was intensified with cobalt chloride and nickel ammonium sulfate (Adams 1981), after which all reacted tissue sections were osmicated and embedded in Araldite.…”

Section: Methodsmentioning

confidence: 99%

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Types of callosally projecting nonpyramidal neurons in rat visual cortex identified by lysosomal HRP retrograde labeling

Hughes

Peters

1992

Anat Embryol

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Callosally projecting neurons, labeled following injection of horseradish peroxidase (HRP) into the 17/18a border of the contralateral hemisphere, have been examined by light and electron microscopy. These neurons exhibit two types of horseradish peroxidase labeling: either a diffuse, Golgi-like labeling, or a granular, punctate labeling. The punctate type of HRP-labeling is the predominant form in nonpyramidal neurons, while pyramidal neurons frequently display either diffuse or punctate labeling. Only punctately labeled neurons have been examined in this study. Light microscopic analyses of 1-microns sections show that in the heavily labeled zone at the area 17/18a border approximately 9% of all of the cells in layer II/III are callosally projecting nonpyramidal cells, and 70% of them are callosally projecting pyramidal cells. Light and electron microscopic examinations indicate that the nonpyramidal neurons are a heterogeneous group which consists of small multipolar neurons, large multipolar neurons, small bipolar neurons, and large bipolar neurons. To investigate the ultrastructural appearance of the punctate HRP labeling, selected neurons have been examined in thin sections. In the electron microscope, the tetramethylbenzidine (TMB) reaction product appears as electron-dense crystals, while the diaminobenzidine (DAB) reaction product appears as dark, electron-dense material which fills the lysosomes. These lysosomes occasionally have a halo of reaction product, but often they are not morphologically distinguishable from dark lysosomes present within neurons from control animals in which the darkening results from staining the thin sections with lead citrate and uranyl acetate. However, labeled neurons possess more dark lysosomes than neurons from control animals. These additional dark lysosomes presumably contain the HRP reaction product visible by light microscopy.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Types of callosally projecting nonpyramidal neurons in rat visual cortex identified by lysosomal HRP retrograde labeling

Hughes

Peters

1992

Anat Embryol

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“…The sections then underwent a 20-minute reaction in the prereaction buffer with 0.3% H 2 O 2 added (Mesulam, 1978;Mesulam et al, 1980;Austyn and Gordon, 1981). This was followed by 15 minutes of incubation with 5% ammonium molybdate in ice-cold sodium acetate buffer to enhance the EM appearance of TMB crystals (Marfurt et al, 1988;Liang and Wan, 1989).…”

Section: Tetramethylbenzidine Visualization Of Ib4-containing Axonsmentioning

confidence: 99%

C‐fiber (Remak) bundles contain both isolectin B4‐binding and calcitonin gene‐related peptide‐positive axons

Murinson

Hoffman

Banihashemi

et al. 2005

J of Comparative Neurology

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Unmyelinated nerve fibers (Remak bundles) in the rodent sciatic nerve typically contain multiple axons. This study asked whether C-fiber bundles contain axons arising from more than one type of neuron. Most small neurons of the lumbar dorsal root ganglion (DRG) are either glial cell line-derived neurotrophic factor dependent or nerve growth factor dependent, binding either isolectin B4 (IB4) or antibodies to calcitonin gene-related peptide (CGRP), respectively. Injection of IB4-conjugated horseradish peroxidase into a lumbar DRG resulted in intense labeling of IB4 axons, with very low background. Visualized by confocal fluorescence, IB4-binding and CGRP-positive nerve fibers originating from different DRG neurons came together and remained closely parallel over long distances, suggesting that these two types of axon occupy the same Remak bundle. With double-labeling immunogold electron microscopy (EM), we confirmed that IB4 and CGRP axons were distinct and were found together in single Remak bundles. Previous studies indicate that some DRG neurons express both CGRP and IB4 binding. To ensure that our immunogold results were not a consequence of coexpression, we studied large populations of unmyelinated axons by using quantitative single-label EM. Tetramethylbenzidine, a chromogen with strong intrinsic signal amplification of IB4-horseradish peroxidase, labeled as many as 52% of unmyelinated axons in the dorsal root. Concomitantly, 97% of the Remak bundles with more than one axon contained at least one IB4-labeled axon. Probabilistic modeling using binomial distribution functions rejected the hypothesis that IB4 axons segregate into IB4-specific bundles (P < 0.00001). We conclude that most Remak bundle Schwann cells simultaneously support diverse axon types with different growth factor dependences.

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“…In rat 6, the segments were cut into 50-m-thick transverse sections on a Vibratome™ (Oxford Laboratories, Oxon, UK). These sections were processed for HRP histochemistry, using tetramethylbenzidine (TMB) as the chromogen, and treated according to a protocol for EM plastic embedding (Marfurt et al, 1988). The sections were inspected under the light microscope, and five sections from each segment were immediately transferred to 1% OsO 4 for 1 hr to stabilize the reaction product, dehydrated in alcohol, and embedded in Durcopan ACM (Fluka Chemie AG, Buchs, Switzerland).…”

Section: Methodsmentioning

confidence: 99%

GABA‐, glycine‐, and glutamate‐immunoreactive bouton profiles in apposition to neurons of the central cervical nucleus in the rat

Ragnarson

Ulfhake

et al. 2002

Anat. Rec.

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The neurons of the central cervical nucleus (CCN) convey information about the position and movements of the head, and receive excitatory input from dorsal neck muscles and the labyrinth. Both of these afferent sources form glutamatergic synaptic contacts with CCN neurons. However, these sensory afferent sources can also inhibit CCN neurons. To further elucidate the synaptic organization, we made an electron microscopic investigation, identifying and evaluating the relative frequency of bouton profiles containing the inhibitory transmitters GABA and glycine in apposition to identified CCN neurons. In addition, labeling for glutamate was performed. The identification of the CCN neurons was made possible by injections of retrograde tracer substances into the cerebellum. These substances were made visible by preembedding immunocytochemistry or postembedding immunogold staining. Such staining was also used to detect the three amino acids that were found in boutons apposed to the identified neurons (cf. Ö rnung et al., J. Comp. Neurol. 1996;365:413-426; Lindå et al., J. Comp. Neurol. 2000;425:10-23). Due to the relatively poor transport of the tracer substances into dendrites of the CCN neurons, the analysis was restricted to the cell body and included bouton profiles in direct apposition to the soma membrane. Data from 10 CCN neurons revealed that about 50% of the apposing bouton profiles were immunoreactive for GABA, and about 34% for glycine. In four neurons, the degree of colocalization of GABA and glycine was determined to be close to 30%. Thus, the vast majority of glycine-labeled profiles also contained GABA, while a considerable fraction of the profiles were immunoreactive for only GABA. The values for glycine immunoreactive bouton profiles presented here may represent somewhat low estimates, depending on the method used. Data from four neurons showed that about 18% of the profiles were labeled for glutamate. The large fraction of purely GABA immunoreactive profiles, or at least a substantial group of them, is suggestive of their derivation from axons descending from the brainstem. Anat Key words: amino acid transmitters; electron microscopy; choleragenoid; horseradish peroxidase; postembedding immunocytochemistryThe central cervical nucleus (CCN) is a spinocerebellarspinovestibular cell group in the upper cervical segments of the spinal cord. It has been studied extensively in the rat and cat (Matsushita and Ikeda, 1975;Wiksten, 1975;Petras, 1977;Cummings and Petras, 1977;Hirai et al., 1978;Wiksten, 1979a, b;Carleton and Carpenter, 1983;Wiksten and Grant, 1983; Hirai et al., 1984a, b;Matsushita and Tanami, 1987;Wiksten, 1987;McKelvey-Briggs et al., 1989;Donevan et al., 1990;Neuhuber et al., 1990;Matsushita, 1991;Matsushita et al., 1991;Popova et al., 1995;Sato et al., 1997). Physiological studies have shown that the CCN transmits information about position and movement of the head, both in space (vestibular input) and in relation to the trunk (neck proprioceptive input) (Hirai et al., 1984b;Popova e...

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Stabilization of tetramethylbenzidine (TMB) reaction product at the electron microscopic level by ammonium molybdate

Cited by 32 publications

References 16 publications

Types of callosally projecting nonpyramidal neurons in rat visual cortex identified by lysosomal HRP retrograde labeling

Types of callosally projecting nonpyramidal neurons in rat visual cortex identified by lysosomal HRP retrograde labeling

C‐fiber (Remak) bundles contain both isolectin B4‐binding and calcitonin gene‐related peptide‐positive axons

GABA‐, glycine‐, and glutamate‐immunoreactive bouton profiles in apposition to neurons of the central cervical nucleus in the rat

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