Microphotometric Determination of Enzyme Activity in Single Cells in Cryostat Sections I. Application of the Gel Film Technique to Microphotometry and Studies on the Intralobular Distribution of Succinate Dehydrogenase and Lactate Dehydrogenase Activities in Rat Liver

Nolte, J.; Pette, D

doi:10.1177/20.8.567

Cited by 85 publications

(31 citation statements)

References 7 publications

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“…Histochemical determination of Ca2 þ -dependent myofibrillar (myosin heavy chain) adenosine triphosphatase (mATPase) and SDH activity were performed on unfixed 10 lm cryostat sections following the modified procedures described previously (Muller and Vogell, 1974;Nolte and Pette, 1972;Quiroz-Rothe and Rivero, 2001). For mATPase staining, sections were incubated at 378C in acetic acid buffer solution (pH ¼ 4.3 or pH ¼ 4.6) containing 1.8 mM CaCl 2 for 5 min or in 2-amino 2-methyl 1-propanol (AMP) buffer solution (pH ¼ 10.4; 36 mM CaCl 2 ) for 14 min.…”

Section: Histology Immunohistochemistry and B-galactosidase Stainingmentioning

confidence: 99%

Insights into function and regulation of small heat shock protein 25 (HSPB1) in a mouse model with targeted gene disruption

Huang

Min

Masters

et al. 2007

Genesis

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The mammalian small heat shock protein (sHSPs) family is comprised of 10 members and includes HSPB1, which is proposed to play an essential role in cellular physiology, acting as a molecular chaperone to regulate diverse cellular processes. Whilst differential roles for sHSPs are suggested for specific tissues, the relative contribution of individual sHSP family members in cellular and organ physiology remains unclear. To address the function of HSPB1 in vivo and determine its tissue-specific expression during development and in the adult, we generated knock-in mice where the coding sequence of hspb1 is replaced by a lacZ reporter gene. Hspb1 expression marks myogenic differentiation with specific expression first confined to developing cardiac muscles and the vascular system, and later in skeletal muscles with specific expression at advanced stages of myoblast differentiation. In the adult, hspb1 expression was observed in other tissues, such as stratified squamous epithelium of skin, oronasal cavity, tongue, esophagus, and uterine cervix but its expression was most prominent in the musculature. Interestingly, in cardiac muscle hsbp1 expression was down-regulated during the neonatal period and maintained to a relatively low steady-level throughout adulthood. Despite this widespread expression, hspb1-/- mice were viable and fertile with no apparent morphological abnormalities in tissues under physiological conditions. However, at the cellular level and under stress conditions (heat challenge), HSPB1 act synergistically with the stress-induced HSPA1 (HSP70) in thermotolerance development, protecting cells from apoptosis. Our data thus indicate a nonessential role for HSPB1 in embryonic development and for maintenance of tissues under physiological conditions, but also shows that it plays an important role by acting synergistically with other HSPs during stress conditions to exert cytoprotection and anti-apoptotic effects.

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Section: Histology Immunohistochemistry and B-galactosidase Stainingmentioning

confidence: 99%

Insights into function and regulation of small heat shock protein 25 (HSPB1) in a mouse model with targeted gene disruption

Huang

Min

Masters

et al. 2007

Genesis

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“…Tissue fixation enables tissue handling and preservation of membranes but at the same time may induce molecular changes in the targets and may pose barriers for penetration of often large immunochemical markers. To avoid fixation of tissue, several techniques have been previously developed, such as the gel film method (Nolte and Pette, 1972) and the semipermeable dialysis membrane technique (McMillan, 1967). These methods, however, have considerable limitations in terms of protein detection procedures.…”

Section: Discussionmentioning

confidence: 99%

In situ blotting: a novel method for direct transfer of native proteins from sectioned tissue to blotting membrane: procedure and some applications.

Okabe

Nyakas

Buwalda³

et al. 1993

J Histochem Cytochem.

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We describe a novel technique for direct transfer of native proteins from unfixed frozen tissues sections to an immobilizing matrix, e.g., nitrocellulose, polyvinyliden difluoride, or positively charged nylon membranes. Proteins are directly blotted onto the membrane, providing optimal accessibility for molecular detection but retaining the anatomic localization at the cellular level. Within 10 min a maximum protein transfer is achieved independent of the protein molecular weight. The total protein bound was 80% of the maximal binding capacity of the blotting membrane and independent of the section thickness. These results indicate that the proteins that bind to the membrane originate from the cut cell monolayer that has direct contact with the blotting membrane. This in situ blotting method provides direct protein mapping from a single cell layer of a tissue section. The procedure includes cryosectioning at 20 microns and collecting sections on a dry blotting membrane at -20 degrees C. For protein transfer the blotted sections are thawed and incubated for 10 min with Tris buffer. After incubation the sections are removed from the membrane by high-pressure spray. The blotted membranes can be subjected to several detection assays. In the present study the presence of several proteins was demonstrated in brain and thymus by immunochemical and enzyme histochemical procedures.

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“…Wider zones of inhibition were found with multifilament sutures than with monofilament sutures in dog and rabbit myocardium (author's unpublished results). Conversely, cells adjacent to absorbing synthetic absorbable sutures showed a considerable increase in dehydrogenasc activity, possibly because of cellular involvement in the metabolism of suture breakdown products and increased phagocytic Succinic dehydrogenase activity can be quantitated in tissue sections.26 27 An incrrase in succinic dehydrogenasc activity is usually accompanied by an increase in both cytochrome oxidase activity and other dehydrogenases.…”

Section: Adenosine Triphosphatase Oxidoreductusesmentioning

confidence: 99%

Cellular enzyme activity at the polymer–tissue interface: A review

Salthouse¹

1976

J. Biomed. Mater. Res.

131

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Microphotometric Determination of Enzyme Activity in Single Cells in Cryostat Sections I. Application of the Gel Film Technique to Microphotometry and Studies on the Intralobular Distribution of Succinate Dehydrogenase and Lactate Dehydrogenase Activities in Rat Liver

Cited by 85 publications

References 7 publications

Insights into function and regulation of small heat shock protein 25 (HSPB1) in a mouse model with targeted gene disruption

Insights into function and regulation of small heat shock protein 25 (HSPB1) in a mouse model with targeted gene disruption

In situ blotting: a novel method for direct transfer of native proteins from sectioned tissue to blotting membrane: procedure and some applications.

Cellular enzyme activity at the polymer–tissue interface: A review

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