Tissue preparation for the demonstration of surface antigens by immunoperoxidase techniques

Judd, M.; Britten, K. J. M.

doi:10.1007/bf01033624

Cited by 20 publications

(4 citation statements)

References 9 publications

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“…Alcohols and acetone are generally very poor at retaining proteins, but conversely do not readily denature protein epitopes 35 , thus making these fixatives ideal for immunohistochemistry [36][37][38] but poor for cartilage GAG histochemistry, unless combined with alcohol. There has been no study of the morphological changes in articular cartilage following alchol fixation.…”

Section: Protein-denaturing Agentsmentioning

confidence: 99%

Histochemical studies of the extracellular matrix of human articular cartilage—a review

Hyllested¹,

Veje²,

Østergaard³

2002

Osteoarthritis and Cartilage

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Section: Protein-denaturing Agentsmentioning

confidence: 99%

Histochemical studies of the extracellular matrix of human articular cartilage—a review

Hyllested¹,

Veje²,

Østergaard³

2002

Osteoarthritis and Cartilage

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“…Synaptophysin is a reliable marker for synaptic vesicles and is used extensively to identify synaptic terminals (Wiedenmann and Franke, 1985;Benson and Cohen, 1996). Weak formaldehyde fixation was used because the standard, e.g., 4% PF, fixation may inhibit immunodetection (Judd and Britten, 1982), especially of receptors (Kirsh and Betz, 1993;Greferath et al, 1995;Nusser and Somogyi, 1997). Various strategies have been attempted to overcome this, including the use of proteinases (Battifora and Kopinski, 1986;Watanabe et al, 1998;Burette et al, 1999;Valtschanoff et al, 1999), microwave irradiation (Bohlhalter et al, 1996), heat (Steward and Halpain, 1999), and higher dilution of fixative (Alvarez et al, 1996).…”

Section: Discussionmentioning

confidence: 99%

Presynaptic kainate receptors in primary afferents to the superficial laminae of the rat spinal cord

Hwang

Pagliardini

Rustioni

et al. 2001

J of Comparative Neurology

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Subunits of glutamate receptors participate in the regulation of sensory transmission at primary afferent synapses in the superficial laminae of dorsal horn (DH). We report here on the distribution of kainate receptors (GluR5/6/7) in these laminae by using light microscope (LM) and electron microscope (EM) immunocytochemistry. Standard (4%) paraformaldehyde fixation resulted in immunostaining for GluR5/6/7 in perikarya and fine processes in lamina II, especially its inner part (IIi). Preembedding EM revealed immunostaining of dendrites, perikarya, and occasional terminals, presumed to be from primary afferent fibers, at the center of glomerular arrangements. In rats perfused with 0.5% paraformaldehyde, LM showed a more punctate staining, mainly in the ventral part of lamina IIi and lamina III, than in material fixed with 4% paraformaldehyde. Approximately two-thirds of GluR5/6/7 puncta were also immunostained with synaptophysin, suggesting that in material fixed with 0.5% paraformaldehyde, a large fraction of these are synaptic terminals. Double immunostained puncta disappear 4 days after dorsal rhizotomy, suggesting that most of GluR5/6/7-immunopositive terminals are from primary afferent fibers. EM material fixed with 0.5% paraformaldehyde confirmed the expression of GluR5/6/7 in numerous synaptic endings with morphology of primary afferents. To determine the type of primary afferent terminals that express GluR5/6/7, two neuroanatomic tracers were injected in the sciatic nerves. The lectin from Bandeiraea simplicifolia (IB4) is selectively taken up by unmyelinated primary afferent fibers that terminate in the outer part of lamina II (IIo) and dorsal part of lamina IIi, whereas the B subunit of the cholera toxin (CTB) is selectively taken up by a broader class of primary afferents which, in superficial DH, terminate mainly in laminae I, ventral part of IIi, and III. Approximately 20% of GluR5/6/7-immunoreactive puncta colocalized with IB4, whereas approximately 40% of GluR5/6/7-immunoreactive puncta colocalized with CTB. The present study shows that (1) GluR5/6/7 does not have a clear and consistent spatial relation with postsynaptic sites, (2) a large number of primary afferents express GluR5/6/7, and (3) these are not limited to one functional class. Thus, modulation by glutamate of primary afferent terminals by means of kainate receptors in the superficial laminae of DH may predominantly involve presynaptic mechanisms.

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“…A major disadvantage of these fixatives is the alteration of tertiary structure by crosslinkage, resulting in alteration of antigenic structures of the tissues (Hancock et al 1982). In contrast, acetone, which precipitates proteins, is a reliable fixative for detection of different markers but morphology is often poor (Judd and Britten 1982). To improve morphology while maintaining staining intensity, we compared the most commonly used mild acetone fixation with pararosaniline.…”

Section: Discussionmentioning

confidence: 99%

Pararosaniline Fixation for Detection of Co-stimulatory Molecules, Cytokines, and Specific Antibody

Schrijver¹,

Melief²,

Meurs³

et al. 2000

J Histochem Cytochem.

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SUMMARYIntegral immunohistochemical analysis of immune responses in frozen sections requires that, in addition to constitutively expressed membrane CD markers, less stable determinants can be reliably visualized. Therefore, we compared the commonly used acetone fixation method with pararosaniline fixation for six determinant categories. These categories included selected constitutively expressed markers, inducible co-stimulatory molecules, pro-and anti-inflammatory cytokines (including the novel cytokine IL-18, also known as IGIF and IL-1 ␥), antigen-specific antibody in plasma cells, bacterial peptidoglycan, and lysosomal acid phosphatase activity. Human spleen and mouse spleen activated by agonistic anti-CD40 antibody or TNP-Ficoll immunization were analyzed in parallel with brain tissue from multiple sclerosis (MS) patients and marmoset monkeys with experimental autoimmune encephalomyelitis (EAE), an animal model for MS. Fixation with pararosaniline resulted in better morphology of all tissues and inhibited endogenous alkaline phosphatase activity in brain tissue. Most determinants could be reliably detected. Staining sensitivity and intensity were markedly increased for selected determinant-tissue combinations, e.g., for IL-4 in human spleen and CD40 in human and mouse spleen. These data show that pararosaniline is a useful alternative to acetone, resulting in superior morphology and specific staining for selected determinant-tissue combinations. This provides additional flexibility for in situ analysis of immune reactivity.

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Tissue preparation for the demonstration of surface antigens by immunoperoxidase techniques

Cited by 20 publications

References 9 publications

Histochemical studies of the extracellular matrix of human articular cartilage—a review

Histochemical studies of the extracellular matrix of human articular cartilage—a review

Presynaptic kainate receptors in primary afferents to the superficial laminae of the rat spinal cord

Pararosaniline Fixation for Detection of Co-stimulatory Molecules, Cytokines, and Specific Antibody

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