A simple, reliable, and sensitive method for nonradioactive in situ hybridization: use of microwave heating to improve hybridization efficiency and preserve tissue morphology.

Lan, Hui‐Yao; Mu, Wei; Ng, Yee Yung; Nikolic-Paterson, David J.; Atkins, Robert C.

doi:10.1177/44.3.8648089

Cited by 88 publications

(44 citation statements)

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“…Detection of mRNA within tissue sections by in situ hybridization used a new microwave-based protocol [18,25]. Formalin-fixed 4-mm paraffin sections were dewaxed, immersed in 0 .…”

Section: Combined In Situ Hybridization and Immunohistochemistry Staimentioning

confidence: 99%

Up-regulation of macrophage colony-stimulating factor (M-CSF) and migration inhibitory factor (MIF) expression and monocyte recruitment during lipid-induced glomerular injury in the exogenous hypercholesterolaemic (ExHC) rat

Miyazaki

Isbel

Lan

et al. 1997

Clinical and Experimental Immunology

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SUMMARYAlthough macrophages play an important role in lipid-induced glomerular injury, we know little of the mechanisms by which hyperlipidaemia induces monocyte recruitment. This study investigated the role of M-CSF and macrophage MIF in monocyte recruitment during the development of lipid-induced glomerular injury in the susceptible ExHC rat strain. Groups of five ExHC rats were fed a high cholesterol diet (HCD) containing 3% cholesterol, 0 . 6% sodium cholate and 15% olive oil, and killed after 3 days, 1, 2 or 6 weeks. Control animals were killed on day 0 or after 6 weeks on a normal diet. Animals were hypercholesterolaemic 3 days after the induction of the HCD, but showed no change in plasma triglycerides over the 6-week period. Glomerular macrophage accumulation was first evident at 1-2 weeks and increased up to week 6, when macrophage-derived foam cells were seen in almost all glomeruli, and segmental lesions and mild proteinuria were also evident. Combined in situ hybridization and immunohistochemistry staining demonstrated that, coincident with the induction of hypercholesterolaemia on day 3, there was marked up-regulation of M-CSF and MIF mRNA expression by intrinsic glomerular cells (mostly mesangial cells and podocytes) which preceded monocyte recruitment. There was a highly significant correlation between the number of M-CSF and MIF-positive cells and glomerular macrophage accumulation over the 6-week period. Although some glomerular macrophages and foam cells exhibited M-CSF and MIF expression, the major source of these molecules was intrinsic glomerular cells. No local macrophage proliferation was observed during the development of glomerular lesions. In conclusion, hypercholesterolaemia caused marked up-regulation of M-CSF and MIF expression by intrinsic glomerular cells, which correlated with monocyte recruitment and the development of lipid-induced glomerular injury. This is the first study to implicate local synthesis of MIF in the pathogenesis of lipid-induced lesions.

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“…Detection of mRNA within tissue sections by in situ hybridization used a new microwave-based protocol [18,25]. Formalin-fixed 4-mm paraffin sections were dewaxed, immersed in 0 .…”

Section: Combined In Situ Hybridization and Immunohistochemistry Staimentioning

confidence: 99%

Up-regulation of macrophage colony-stimulating factor (M-CSF) and migration inhibitory factor (MIF) expression and monocyte recruitment during lipid-induced glomerular injury in the exogenous hypercholesterolaemic (ExHC) rat

Miyazaki

Isbel

Lan

et al. 1997

Clinical and Experimental Immunology

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“…For double labeling, brain sections were sequentially stained with nNOS and citrulline antisera. After completing staining with the first antiserum (citrulline), sections were microwaved as described (24). The first primary was visualized with the Vectastain ABC Elite Kit.…”

Section: Methodsmentioning

confidence: 99%

Neuronal nitric oxide synthase alternatively spliced forms: Prominent functional localizations in the brain

Eliasson

Blackshaw

Schell

et al. 1997

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

262

219

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Neuronal nitric-oxide synthase (nNOS) is subject to alternative splicing. In mice with targeted deletions of exon 2 (nNOS ⌬͞⌬ ), two alternatively spliced forms, nNOS␤ and ␥, which lack exon 2, have been described. We have compared localizations of native nNOS␣ and nNOS␤ and ␥ by in situ hybridization and immunohistochemistry in wild-type and nNOS ⌬͞⌬ mice. To assess nNOS catalytic activity in intact animals we localized citrulline, which is formed stoichiometrically with NO, by immunohistochemistry. nNOS␤ is prominent in several brain regions of wild-type animals and shows 2-to 3-fold up-regulation in the cortex and striatum of nNOS ⌬͞⌬ animals. The persistence of much nNOS mRNA and protein, and distinct citrulline immunoreactivity (cit-IR) in the ventral cochlear nuclei and some cit-IR in the striatum and lateral tegmental nuclei, indicate that nNOS␤ is a major functional form of the enzyme in these regions. Thus, nNOS␤, and possibly other uncharacterized splice forms, appear to be important physiological sources of NO in discrete brain regions and may account for the relatively modest level of impairment in nNOS ⌬͞⌬ animals.Nitric oxide (NO), formed by neuronal nitric-oxide synthase (nNOS) (1), has major signaling functions in the central and peripheral nervous system (2, 3). Alternative splicing of the nNOS gene is prominent with more than 10 differently spliced nNOS transcripts reported (4-10). The principal, exon 2-containing, form of nNOS (nNOS␣) accounts for the great majority of catalytic activity in the brain, as mice with targeted deletions of exon 2 (nNOS ⌬͞⌬ ) display an Ϸ95% reduction in NOS catalytic activity (11). Though NO has been implicated in development (12) and synaptic plasticity (13), these mice appear grossly normal, lack obvious histopathological abnormalities in the central nervous system, and reproduce effectively (11), suggesting that the residual NOS activity might protect the nNOS ⌬͞⌬ mutant mice from serious pathology. Two alternatively spliced forms of nNOS, ␤ and ␥, that lack exon 2 and thus persist in the nNOS ⌬͞⌬ mice, have a regional distribution that parallels the pattern of the residual activity (4).The human nNOS gene contains 29 exons of which all but exon 1 are translated to generate nNOS␣, a 150-kDa protein (1, 8). nNOS␤ and ␥, as described in mice, are generated by alternative splicing that skips exon 2 and employs different first exons, 1a and 1b, respectively (4) (Fig. 1). Translation of nNOS␤ is initiated at a CTG-initiation codon within exon 1a generating a 136-kDa NOS protein with six unique N-terminal amino acids. Translation of nNOS␥ is initiated at an ATGinitiation codon within exon 5 generating a truncated, 125-kDa form of nNOS␣. Both nNOS␤ and nNOS␥ lack the PSD-95͞ discs large͞ZO-1 homology domain (PDZ) domain that is localized within exon 2. This domain, also referred to as DHR or GLGF, mediates association of nNOS␣ with postsynaptic density protein 95 ref. 4). PSD-95 appears to anchor nNOS␣ to neuronal membranes in the vicinity of the Nmethyl-D-as...

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“…The MW heating method was also used for enhancement of mRNA in situ hybridization (Lan et al 1996;McMahon and McQuaid 1996;Sibony et al 1995) and for the TUNEL method with the use of short MW heating for 1 min (Strater et al 1995).…”

Section: Demonstrated Utility Of Ar-ihc In Analytical Morphologymentioning

confidence: 99%

Antigen Retrieval Immunohistochemistry: Past, Present, and Future

Shi

Côté²,

Taylor

1997

J Histochem Cytochem.

478

326

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SUMMARYThe antigen retrieval (AR) technique, which is predominantly based on hightemperature heating of tissues, is used as a non-enzymatic pretreatment for immunohistochemical staining of formalin-fixed, paraffin-embedded tissue sections. It has been widely applied in pathology and analytical morphology. The existence of a growing body of literature on the AR technique raises a number of interesting issues for the further development of AR. These issues include the use of a "test battery" and the concept of "maximal retrieval" applied to the selection of optimal test protocols for the standardization of AR. (J Histochem Cytochem 45:327-343, 1997)

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A simple, reliable, and sensitive method for nonradioactive in situ hybridization: use of microwave heating to improve hybridization efficiency and preserve tissue morphology.

Cited by 88 publications

References 0 publications

Up-regulation of macrophage colony-stimulating factor (M-CSF) and migration inhibitory factor (MIF) expression and monocyte recruitment during lipid-induced glomerular injury in the exogenous hypercholesterolaemic (ExHC) rat

Up-regulation of macrophage colony-stimulating factor (M-CSF) and migration inhibitory factor (MIF) expression and monocyte recruitment during lipid-induced glomerular injury in the exogenous hypercholesterolaemic (ExHC) rat

Neuronal nitric oxide synthase alternatively spliced forms: Prominent functional localizations in the brain

Antigen Retrieval Immunohistochemistry: Past, Present, and Future

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