The use of propane/isopentane mixtures for rapid freezing of biological specimens

Vichows Archiv A Pathol Anat

Terada

Fujii

et al. 1996

Morphological studies using immersion or perfusion fixation methods do not reveal the ultrastructure of functioning kidneys with normal circulation. A simple apparatus was developed for freezing the kidneys in vivo without stopping the blood supply, and the ultrastructure of the glomerular capillary loops was examined under different haemodynamic conditions. Mouse kidneys were frozen under normal blood flow conditions; others were frozen in the same way after ligation of the abdominal aorta at a point caudal to the renal arteries. They were then processed for the freeze-substitution or deep-etching method. Good ultrastructural preservation was obtained within about 5 microM depth from the frozen tissue surface. Functioning glomeruli with normal blood flow possessed open capillary lumens, different shapes of foot processes and atypical basement membranes with low density. Moreover, heterogeneity in width between foot processes was identified on the replica membranes. Under the acute conditions used to increase blood supply into the kidneys, the spaces between the flat foot processes became more widely dilated and the basement membrane was seen to be three-layered. The ultrastructure of glomeruli in functioning kidneys has been demonstrated for the first time by this "in vivo cryotechnique."

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Dynamic structure of glomerular capillary loop as revealed by an in vivo cryotechnique

Vichows Archiv A Pathol Anat

Terada

Fujii

et al. 1996

“…Roots and leaves from 20-d-old plants of L. esculentum (wild type, mutant, and mutant + NA) and P. vulgaris (control and Fe-loaded) were cut to 1-to 2-mm pieces, rapidly frozen in a 2:l mixture of pr0pane:isopentane at -196°C (Jehl et al, 1981), freeze-dried at -5O"C, and stored over silica gel. Root tissue was taken from approximately 1-cm-long root tips and dista1 zones of lateral root formation.…”

Section: Edaxmentioning

confidence: 99%

Subcellular Localization and Characterization of Excessive Iron in the Nicotianamine-less Tomato Mutant chloronerva

1995

To understand the function of the Fe2+-complexing compound nicotianamine (NA) in the iron metabolism of plants we have localized iron and other elements in the NA-containing tomato wild type (Lycopersicon esculentum) and its NA-free mutant chloronerva by quantitative x-ray microanalysis. Comparison of element composition of the rhizodermal cell walls indicated that the wild type accumulated considerable amounts of iron and phosphorus in the cell wall, whereas in the mutant iron and phosphorus were detected in the cytoplasm and vacuoles of the rhizodermis. In mutant leaves containing high iron concentrations in the symplast, electron-dense inclusions were detected in chloroplasts and phloem. Such particles, consisting mainly of iron and phosphorus, were never found in the wild type and were very rarely detected in young chlorotic mutant leaves or after treatment of the mutant with NA. For further characterization the electron-dense inclusions in mutant leaves were isolated and compared by sodium dodecyl sulfate-gel electrophoresis and immunoblotting to ferritin from iron-loaded Phaseolus vulgaris leaves. Antibodies raised against purified Phaseolus leaf ferritin were used. Neither in mutant nor in wild type (iron loaded and control) was ferritin protein detected. These results suggest that the electron-dense inclusions in mutant leaves are not identical with ferritin. It is concluded that NA is necessary to complex ferrous iron in a soluble and available form within the cells. In the absence of NA the precipitation of excessive iron in the form of insoluble ferric phosphate compounds could protect the cells from iron overload.

“…Isopentane-propane cryogen (-193°C), which has been prepared by bubbling propane gas in liquid isopentane cooled down in liquid nitrogen, is simultaneously poured onto the cryocut organs, followed by liquid nitrogen. The volume ratio of isopentane to liquidized propane is usually between 1:2 and 1:3 to achieve the lowest temperature (-193°C) during the QF method [9]. Thereafter, the frozen organs are removed by a dental electric drill in liquid nitrogen, and some well-frozen pieces of the removed sample tissues are processed for the next sample preparation step.…”

Section: How To Perform In Vivo Cryotechniquementioning

confidence: 99%

Advanced Application of the In Vivo Cryotechnique to Immunohistochemistry for Animal Organs

Terada

Acta Histochem. Cytochem.

2004

The quick-freezing method often used for physical fixation also contributed enormously to the advancement of morphology, but it could not provide enough information about the dynamic morphological changes in vivo in living animal organs. Therefore, we developed the in vivo cryotechnique in 1995, which could directly cryofix organs in vivo under anesthetized conditions without stopping blood circulation or producing effects of anoxia. We have already reported new findings about the in vivo ultrastructures of living animal organs with the cryotechnique followed by freeze-substitution or replica preparation. Recently, it has also been applied to other morphological analyses in vivo, including immunohistochemistry and FISH, for obtaining dynamically functioning structures of cells and tissues at a light microscopic level. In such experimental processes, the in vivo cryotechnique has been shown to have the added benefit of direct antigen-retrieval effects since it reduces several steps of retrieval treatments which are required in common paraffin-embedded samples prepared by the conventional fixation and dehydration. In conclusion, the in vivo cryotechnique allows us to investigate the functioning real morphology of living animals, which was technically difficult to be observed before, and perform dynamic immunohistochemistry of cells and tissues in various animal organs in vivo at ultimate time-resolution.