Modified formalin and methanol fixation methods for molecular biological and morphological analyses

Noguchi, Masayuki; Furuya, J Shuichiroh; Takeuchi, Tomoyoshi; Hirohashi, Setsuo

doi:10.1111/j.1440-1827.1997.tb04442.x

Cited by 88 publications

(62 citation statements)

References 13 publications

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“…Another group of bAECs was maintained in basal medium without FITC-insulin as a control. Cells were then fixed with cold methanol for 10 min at Ϫ20°C before immunocytochemical staining (see below) (18).…”

Section: Methodsmentioning

confidence: 99%

The vascular endothelial cell mediates insulin transport into skeletal muscle

Wang¹,

Liu²,

Li³

et al. 2006

American Journal of Physiology-Endocrinology and Metabolism

123

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The pathways by which insulin exits the vasculature to muscle interstitium have not been characterized. In the present study, we infused FITC-labeled insulin to trace morphologically (using confocal immunohistochemical methods) insulin transport into rat skeletal muscle. We biopsied rectus muscle at 0, 10, 30, and 60 min after beginning a continuous (10 mU·min−1·kg−1), intravenous FITC-insulin infusion (with euglycemia maintained). The FITC-insulin distribution was compared with that of insulin receptors (IR), IGF-I receptors (IGF-IR), and caveolin-1 (a protein marker for caveolae) in skeletal muscle vasculature. We observed that muscle endothelium stained strongly for FITC-insulin within 10 min, and this persisted to 60 min. Endothelium stained more strongly for FITC-insulin than any other cellular elements in muscle. IR, IGF-IR, and caveolin-1 were also detected immunohistochemically in muscle endothelial cells. We further compared their intracellular distribution with that of FITC-insulin in cultured bovine aortic endothelial cells (bAECs). Considerable colocalization of IR or IGF-IR with FITC-insulin was noted. There was some but less overlap of IR or IGF-IR or FITC-insulin with caveolin-1. Immunoprecipitation of IR coprecipitated caveolin-1, and conversely the precipitation of caveolin-1 brought down IR. Furthermore, insulin increased the tyrosine phosphorylation of caveolin-1, and filipin (which inhibits caveolae formation) blocked insulin uptake. Finally, the ability of insulin, IGF-I, and IGF-I-blocking antibody to diminish insulin transport across bAECs grown on transwell plates suggested that IGF-IR, in addition to IR, can also mediate transendothelial insulin transit. We conclude that in vivo endothelial cells rapidly take up and concentrate insulin relative to plasma and muscle interstitium and that IGF-IR, like IR, may mediate insulin transit through endothelial cells in a process involving caveolae.

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Section: Methodsmentioning

confidence: 99%

The vascular endothelial cell mediates insulin transport into skeletal muscle

Wang¹,

Liu²,

Li³

et al. 2006

American Journal of Physiology-Endocrinology and Metabolism

123

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“…Preservation in methanol and propanol is contraindicated [38], although tissue samples preserved in 100% methanol will yield higher molecular weight DNA than samples preserved in formalin [63].…”

Section: Fluid-preserved Samplesmentioning

confidence: 99%

Obtaining, Storing and Archiving Specimens and Tissue Samples for Use in Molecular Studies

Prendini¹,

Hanner²,

DeSalle³

2002

Techniques in Molecular Systematics and Evolution

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“…Samples from 39 patients with well-differentiated adenocarcinomas were embedded in paraffin for LCM after their fixation in methanol for 24 h, as described elsewhere (Noguchi et al, 1997). From the other 20 patients, we obtained paired samples from cancerous and adjacent non-cancerous tissues, which were frozen immediately in liquid nitrogen and stored at À801C until required.…”

Section: Cell Lines and Primary Tumorsmentioning

confidence: 99%

Genomic loss and epigenetic silencing of very-low-density lipoprotein receptor involved in gastric carcinogenesis

et al. 2006

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Homozygous loss in the genomic sequence, a mechanism for inactivating tumor-suppressor genes (TSGs) in cancer, has been used as a tag for the identification of novel TSGs, and array-based comparative genomic hybridization (array-CGH) has a great potential for high-throughput identification of this change. We identified a homozygous loss of the very-low-density lipoprotein receptor (VLDLR) gene (9p24.2) from genome-wide screening for copynumber alterations in 32 gastric cancer (GC) cell lines using array-CGH. Although previous reports demonstrated mRNA or protein expression of VLDLR in various cancers including GC, the association between genomic losses or epigenetic silencing of this gene and carcinogenesis has never been reported before. Homozygous deletion of VLDLR was also seen in primary GCs, albeit infrequently, and about half of GC cell lines showed lost or reduced VLDLR expression. The VLDLR expression was restored in gene-silenced GC cells after treatment with 5-aza 2 0 -deoxycytidine. According to methylation analyses, hypermethylation of the VLDLR promoter region, which all of GC lines without its expression showed, occurred in some primary GCs. Restoration of VLDLR type I expression in GC cells reduced colony formation. These results suggest that not only the expression of VLDLR but also genetic or epigenetic silencing of this gene may contribute to tumor formation and be involved in gastric carcinogenesis.

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Modified formalin and methanol fixation methods for molecular biological and morphological analyses

Cited by 88 publications

References 13 publications

The vascular endothelial cell mediates insulin transport into skeletal muscle

The vascular endothelial cell mediates insulin transport into skeletal muscle

Obtaining, Storing and Archiving Specimens and Tissue Samples for Use in Molecular Studies

Genomic loss and epigenetic silencing of very-low-density lipoprotein receptor involved in gastric carcinogenesis

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