Microwave Staining of Enteric Neurons Using Cuprolinic Blue (Quinolinic Phthalocyanin) Combined with Enzyme Histochemistry and Peroxidase Immunohistochemistry

Ginneken, Chris Van; Smet, Marcel J. De; Meir, Frans J. Van; Weyns, A.

doi:10.1177/002215549904700103

Cited by 8 publications

(6 citation statements)

References 39 publications

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“…This method was proposed for the "specific, selective and complete" visualisation of neurons (Heinicke et al 1987;Karaosmanoglu et al 1996;Van Ginneken et al 1999;Román et al 2001). Although our present data are not directly comparable to those of an earlier study based on silver impregnated material (Brehmer and Stach 1998), both the total number of neurons demonstrated and the proportions of morphologically identified neurons are in the same range.…”

Section: Discussioncontrasting

confidence: 96%

Morphological phenotyping of enteric neurons using neurofilament immunohistochemistry renders chemical phenotyping more precise in porcine ileum

Brehmer

Schrödl

Neuhuber

2002

Histochem Cell Biol

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The aim of the study was: (1) to test the suitability of neurofilament (NF) immunohistochemistry for representing the shapes of morphologically defined neuron types in the pig ileum myenteric plexus, (2) to estimate the proportions of these neuron types as related to the whole myenteric neuron population and (3) to demonstrate the usefulness of a refined morphological classification of enteric neurons on the paradigm of calcitonin gene-related peptide (CGRP)-immunoreactive neurons. So far, immunoreactivity for this peptide was supposed to be present in the pig enteric nervous system only in type II neurons. Ileal whole mounts of two pigs were stained with the cuprolinic blue (CB) method and, thereafter, incubated with an antibody pool against NF proteins (70, 160 and 210 kDa), visualised with a fluorochrome-tagged secondary antibody. The structural representation of morphologically defined myenteric neuron types typical for pig ileum (Stach I, II, IV, V and VI) was equivalent to their silver impregnated image, as demonstrated in previous studies. Counts of CB-stained neurons revealed between 2,526 and 2,662 neurons per square centimetre in one pig and between 2,027 and 2,763 in the other. As related to these total neuron numbers, the proportions of type I neurons were 1.7% and 1.5%, of type II neurons 7.2% and 7.9%, of type IV neurons 1.9% and 2.4%, of type V neurons 1.1% and 1.5%, and of type VI neurons 1.3% each. These values are generally comparable with those estimated earlier on silver impregnated material. Double labelling for NF and CGRP indicated that CGRP-immunoreactive smooth contoured neurons with long processes could be subdivided into two distinct morphological neuron types, i.e. type II and type V. We conclude that NF immunohistochemistry is an appropriate tool for representation of morphologically defined enteric neuron types in the pig. Combination of this technique with immunohistochemistry for neuroactive substances may be useful for making both morphological and chemical classification schemes mutually more precise.

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Section: Discussioncontrasting

confidence: 96%

Morphological phenotyping of enteric neurons using neurofilament immunohistochemistry renders chemical phenotyping more precise in porcine ileum

Brehmer

Schrödl

Neuhuber

2002

Histochem Cell Biol

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“…Glial cells were recognized by their intensely S100β-stained soma (arrows). Bar 50 μm substance (Holst and Powley 1995;Van Ginneken et al 1999) and is considered to be equivalent to anti-HuC/D for enteric neuronal quantification (Phillips et al 2004;Ganns et al 2006;Murphy et al 2007). Based on our counts, HuC/D and CB display a similar efficiency in labeling myenteric neurons (89.5% and 93.5%, respectively), reflecting immunofluorescence in whole-mount preparations (96.3% and 95.3%, respectively; Ganns et al 2006).…”

Section: Discussionmentioning

confidence: 99%

“…Based on our counts, HuC/D and CB display a similar efficiency in labeling myenteric neurons (89.5% and 93.5%, respectively), reflecting immunofluorescence in whole-mount preparations (96.3% and 95.3%, respectively; Ganns et al 2006). Although CB has been proposed as an effective counterstain of enteric neurons for peroxidase immunocytochemistry (Holst and Powley 1995;Van Ginneken et al 1999), it is difficult to combine with immunohistochemical processing (Murphy et al 2007) because of the occurrence of staining problems such as light neuronal perikaryon staining and high background. In agreement with this, our data indicate that HuC/D immunostaining is more effective than CB in outlining neuronal cell bodies in terms of sharpness, intensity, and image definition, together with a lack of background.…”

Section: Discussionmentioning

confidence: 99%

Quantitative evaluation of myenteric ganglion cells in normal human left colon: implications for histopathological analysis

et al. 2009

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The analysis of myenteric neurons is becoming increasingly important for the assessment of enteric nervous system injury and degeneration occurring in motor disorders of the gut. Limited information is presently available on the quantitative estimation of myenteric neurons and glial cells in paraffin-embedded colonic sections; additional data would be useful for diagnostic purposes. In this morphometric study, we performed immunohistochemistry to count myenteric neurons and glial cells in paraffin sections of human colon. Serial cross sections of formalin-fixed paraffin-embedded full-thickness normal human left colon (n = 10, age-range: 50-72 years) were examined. HuC/D and S100beta antigens were found to be the best markers for the detection of neurons and glial cells, respectively. Significant correlations were noted between the numbers of neurons/glial cells and the respective myenteric ganglion areas. These findings suggest that HuC/D-S100beta-immunostained paraffin cross sections of human colon can be regarded as valuable tools for the quantitative estimation of myenteric neurons and glial cells. Based on the present method, only a limited number of paraffin sections are needed for reliable quantitative assessments of myenteric ganglion cells, thus allowing fast and simple approaches in the settings of the histopathological diagnosis of colonic motility disorders and retrospective evaluations of pathological archival tissue specimens.

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“…On the other hand, NF immunostaining yielded dense immunoreactivity in the processes and in the ganglionic neuropil; the neuronal outlines were therefore not clear and counting was more difficult. Double labelling with the two markers (Holst and Powley 1995;Van Ginneken et al 1999) made the localisation of NF positivity more accurate and allowed a morphological classification of the NF-stained neurons. Those shapes with an average area of 350 µm 2 corresponded most closely to the type I and III neurons, but rarely to the type II neurons originally described by Dogiel (1899).…”

Section: Discussionmentioning

confidence: 99%

Evaluation of the total number of myenteric neurons in the developing chicken gut using cuprolinic blue histochemical staining and neurofilament immunocytochemistry

Román

Krecsmarik

Bagyánszki

et al. 2001

Histochem Cell Biol

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The aim of this study was to find an improved method with which to stain the entire population of myenteric neurons in the different segments of the developing chicken intestine. Histochemical staining with cuprolinic blue (quinolinic phthalocyanine) and immunostaining against neurofilament (NF) were performed on whole mounts prepared from intestinal segments of embryonic (day 19 of incubation) and hatched (1, 2, 4 and 7 days after hatching) chickens. Double labelling was performed to evaluate to what extent the two markers visualise the same nerve cell population. Cuprolinic blue stained neuronal somata highly selectively, whereas processes and glia cells were poorly labelled. The cuprolinic blue-positive neurons were uniform in shape. NF immunostaining revealed a morphologically highly variable neuron population. Double labelling with cuprolinic blue and NF resulted in an intensification of both stainings, allowing an accurate morphological classification of NF-stained myenteric neurons. Data obtained from the counting of cuprolinic blue-positive neurons were subjected to two-way ANOVA and the Tukey probe. The densities of ganglia and neurons were found to decrease, and the mean number of neurons per myenteric ganglion to increase, with different dynamics along the longitudinal axis of the gut during the examined time span. The variances in the number of NF-positive neurons were not homogeneous, and the data were therefore not suitable for ANOVA. Accordingly, only semiquantitative conclusions could be drawn.

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Microwave Staining of Enteric Neurons Using Cuprolinic Blue (Quinolinic Phthalocyanin) Combined with Enzyme Histochemistry and Peroxidase Immunohistochemistry

Cited by 8 publications

References 39 publications

Morphological phenotyping of enteric neurons using neurofilament immunohistochemistry renders chemical phenotyping more precise in porcine ileum

Morphological phenotyping of enteric neurons using neurofilament immunohistochemistry renders chemical phenotyping more precise in porcine ileum

Quantitative evaluation of myenteric ganglion cells in normal human left colon: implications for histopathological analysis

Evaluation of the total number of myenteric neurons in the developing chicken gut using cuprolinic blue histochemical staining and neurofilament immunocytochemistry

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