Quantitative immunocytochemistry using an image analyzer. I. Hardware evaluation, image processing, and data analysis

Mize, R. Ranney; Holdefer, Robert N.; Nabors, L. Burt

doi:10.1016/0165-0270(88)90125-2

Cited by 110 publications

(46 citation statements)

References 37 publications

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“…It is capable of the wide range of image processing and analysis functions required for fibre analysis. [17][18][19][20] Linear and geometric measurements including diameter, length, area, and perimeter of fibres can be made. Up to 500 measurements can be stored at one time in the results window.…”

Section: Morphometric Analysismentioning

confidence: 99%

Validation of a simple, rapid, and economical technique for distinguishing type 1 and 2 fibres in fixed and frozen skeletal muscle

Behan

Cossar²,

Madden³

et al. 2002

Journal of Clinical Pathology

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Aims: To produce a method of distinguishing between type 1 and 2 skeletal muscle fibres that would be more economical and reproducible than the standard ATPase method and be applicable to both fixed and frozen tissue. Because the ATPase method has been accepted as the basis for fibre identification for the past 50 years, the new method should not give significantly different results. Methods: Isoforms of myosin correlate with isoforms of myofibrillar ATPase and an immunohistochemical (IHC) double labelling protocol was devised using monoclonal antibodies to fast and slow myosin. This required one tissue section rather than four. The results of the two methods were compared by means of morphometric analysis of skeletal muscle biopsies from 20 normal healthy volunteers. Results: There were no significant differences (p = 0.57) in the percentages of type 1 (46% using the IHC method v 48% using ATPase) or type 2 fibres (54% v 52%, respectively). The 2a and 2b subtypes were distinguished easily. Analysis of variance revealed that cross sectional area (µm 2 ), diameter (µm), form factor, and density of fibre staining (a measure of substrate-enzyme or protein) were all similar. The method worked equally well on fixed material. Conclusion: An IHC method based on the fast and slow isoforms of myosin shows no significant differences in fibre type analysis from the standard ATPase method although it provides important advantages because it is applicable to fixed (including archival) material, is economical and reproducible, and yields a permanent preparation.

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Section: Morphometric Analysismentioning

confidence: 99%

Validation of a simple, rapid, and economical technique for distinguishing type 1 and 2 fibres in fixed and frozen skeletal muscle

Behan

Cossar²,

Madden³

et al. 2002

Journal of Clinical Pathology

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“…Quantitative analysis of the extent of TH-positive fiber staining within the striatum was based on a segmented field approach (Mize et al, 1988;Burke et al, 1990). This analysis was restricted to four animals lesioned at PND14 and six animals lesioned at PND28.…”

Section: Methodsmentioning

confidence: 99%

Early Developmental Destruction of Terminals in the Striatal Target Induces Apoptosis in Dopamine Neurons of the Substantia Nigra

et al. 1997

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Many developing neural systems with peripheral projections depend on their target for trophic support during a critical period of natural cell death. Much less is known about central systems. That dopaminergic neurons of the substantia nigra may depend on their target, the striatum, during development is suggested by the presence of a natural apoptotic cell death event in these neurons that can be augmented by an early developmental axon-sparing striatal injury. To further assess the target dependence of these neurons, we have used the selective neurotoxin 6-hydroxydopamine to lesion their terminals within the striatum during development, while sparing intrinsic striatal target neurons. This lesion results in an induction of apoptotic cell death in phenotypically defined dopaminergic neurons that appears on the third postlesion day and persists until the tenth. This inducibility of cell death is dependent on developmental age: it is most marked before postnatal day (PND) 14. As late as PND42, inducibility is still detectable but much less so. In addition, at day 42 the morphology of cell death changes and becomes nonapoptotic in some cells. We conclude that terminal injury during a critical period of postnatal development, like axon-sparing target injury, induces augmented apoptotic death in mesencephalic dopaminergic neurons. These results suggest that these neurons have a period of target dependence. Regulation of this dependence is likely to influence the mature adult number of dopaminergic neurons.

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“…The sections were treated with 1% borohydride/PB, washed repeatedly in phosphate-buffered saline, incubated overnight in the anti-ChAT antibody (Boehringer Mannheim, 1:100 dilution in phosphate-buffered saline), and further processed for EM studies as described (17 (18), to perform the quantification. With this method, detected fibers are extracted from background and reduced to a single pixel in width; total number ofpixels counted per area, therefore, represents total fiber length (i.e., total fiber network per area) (18). ChAT-IR profiles were measured in all cortical layers adjacent to the lesion site-specifically, within area 4 of the somatosensory rat cortex at the mid-basalis level (19).…”

Section: Methodsmentioning

confidence: 99%

Nerve growth factor-induced synaptogenesis and hypertrophy of cortical cholinergic terminals.

Garofalo

Ribeiro‐da‐Silva

Cuello

1992

Proc. Natl. Acad. Sci. U.S.A.

135

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In this study light and EM quantitative analysis were used to examine whether exogenous nerve growth factor (NGF) could affect terminal fields and synaptic connections in the adult rat brain in vivo. Adult rats received, immediately after unilateral decortication, 2.5S NGF (12 lag/day) or vehicle intracerebroventricularly for 7 days.Thirty days after the lesion cholinergic fiber length was quantified, using image analysis, in the remaining cortical area adjacent to the lesion site in each animal. Rats that had received vehicle showed a significantly reduced cortical choline acetyltransferase-immunoreactive fiber network in the remaining cortex when compared with control animals. By contrast, the network in lesioned rats that had received 2.5S NGF was not different from control animals. Furthermore, the number of cortical choline acetyltransferase-immunoreactive varicosities, which decreased in vehicle-treated lesioned rats, significantly increased above control in lesioned rats that had received 2.5S NGF. At the ultrastructural level, 30 days after the lesion, animals that had received vehicle showed shrunken cholinergic boutons in cortical layer V and fewer synapses compared with control animals. Exogenous NGF, administered to lesioned rats, increased to supernormal levels both size of cholinergic boutons and number of synaptic contacts. These parameters were unaltered in unlesioned rats treated with NGF. This study demonstrates that exogenous NGF can cause significant compensatory changes in terminal fields and synaptic connections in the adult fully differentiated central nervous system.Repairing and restoring function to injured neural pathways is the ultimate goal of regeneration studies in the central nervous system (CNS). Achieving this goal, though, requires that some degree of reorganization in neural circuitry and connections should occur, but presently little is known about agents that can regulate such events in the fully differentiated CNS. Potential candidates to mediate these phenomena are growth factors, of which nerve growth factor (NGF) is a prototype (1). Numerous studies have now demonstrated the neurotrophic effects of NGF on adult basal forebrain cholinergic neurons in vivo (2-5). However, despite these studies there is no direct morphological evidence to indicate that NGF actions can be extended to CNS synaptic remodeling. For the most part, studies involving exogenous NGF and brain injury have examined effects of NGF on cholinergic neurochemical markers, cell survival, and morphology and have provided only indirect evidence for sprouting (6). Although NGF can facilitate axonal growth when a bridge of peripheral nervous tissue is provided (7), the full extent of trophic factor-induced effects in the mature CNS remains to be established. In particular, whether these substances can modify synapses in the adult animal is unknown. We have, therefore, investigated whether exogenous NGF can affect these elements in the central cholinergic system.The nucleus basalis magnocellularis (NBM) ...

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Quantitative immunocytochemistry using an image analyzer. I. Hardware evaluation, image processing, and data analysis

Cited by 110 publications

References 37 publications

Validation of a simple, rapid, and economical technique for distinguishing type 1 and 2 fibres in fixed and frozen skeletal muscle

Validation of a simple, rapid, and economical technique for distinguishing type 1 and 2 fibres in fixed and frozen skeletal muscle

Early Developmental Destruction of Terminals in the Striatal Target Induces Apoptosis in Dopamine Neurons of the Substantia Nigra

Nerve growth factor-induced synaptogenesis and hypertrophy of cortical cholinergic terminals.

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