Air-Inflation of Murine Lungs with Vascular Perfusion-Fixation

10.3791/62215 ◽  
2021 ◽  
Author(s):  
Stacey M. Thomas ◽  
Joseph Bednarek ◽  
William J. Janssen ◽  
Patrick S. Hume
Keyword(s):  
Vascular Perfusion ◽  
Perfusion Fixation

Perfusion fixation of lungs for structure-function analysis: credits and limitations

1982 ◽  
Vol 53 (2) ◽  
pp. 528-533 ◽  
Author(s):  
H. Bachofen ◽  
A. Ammann ◽  
D. Wangensteen ◽  
E. R. Weibel
Keyword(s):  
Osmium Tetroxide ◽  
Function Analysis ◽  
Transpulmonary Pressure ◽  
Uranyl Acetate ◽  
Vascular Perfusion ◽  
Lung Weight ◽  
Physiological Variables ◽  
Isotonic Buffer ◽  
Perfusion Fixation

The quality of tissue preservation in lungs fixed by vascular perfusion has been reevaluated. Excised rabbit lungs inflated to 60% of total lung capacity were perfused (zone III conditions) with different but widely used fixatives. The effects of the perfusates on pertinent physiological variables have been assessed by a continuous monitoring, the effects on the pulmonary microstructure by qualitative and morphometric analysis of electron micrographs. Important results include the following. 1) Perfusions with isotonic glutaraldehyde at flow rates within the physiological range produce large increases of perfusion pressure and lung weight that reflect intracellular, interstitial, and intra-alveolar edema. 2) No edema occurs if glutaraldehyde is added to isotonic buffer solutions (total osmolarity 510 mosM). 3) Glutaraldehyde as sole perfusate does not fully eliminate the retractive force of lung tissue. Upon release of transpulmonary pressure the lungs retract by an indeterminable amount. 4) Satisfactory results can be obtained by sequential perfusion with osmium tetroxide and uranyl acetate or glutaraldehyde (510 mosM) followed by osmium tetroxide and uranyl acetate. The latter combination yields optimal preparations to study the alveolar and capillary architecture but causes a hyperosmotic volume loss of lung cells (cell shrinkage).

Temperature-Related Ultrastructural Changes in Early Stage Amelogenesis in Vascularly Perfused Rat Incisors

1987 ◽  
Vol 1 (2) ◽  
pp. 371-379 ◽  
Author(s):  
Y. Takano ◽  
M. Akai ◽  
S. Matsuo ◽  
S. Wakisaka ◽  
H. Ichikawa ◽  
...  
Keyword(s):  
Granular Material ◽  
Matrix Protein ◽  
Early Stage ◽  
Physiological Solution ◽  
Ultrastructural Changes ◽  
Vascular Perfusion ◽  
Coated Pits ◽  
The Matrix ◽  
Perfusion Fixation ◽  
Definition Of

Vascularly perfused rat incisors were investigated by electron microscopy in order to achieve further definition of the origin and nature of stippled material in developing enamel. A prolonged (20-minute) pre-wash perfusion of the rat with a physiological solution at 37°C prior to perfusion fixation retained good morphology of the secretory ameloblasts and adjacent enamel, showing virtually no extracellular accumulation of granular material. Granular material appeared throughout the developing enamel after pre-wash perfusion at low temperatures (0-2°C), and accumulated in the extensively dilated extracellular spaces between the proximal portions of Tomes' processes, where numerous coated pits and coated vesicles were located. Vascular perfusion at 37°C, preceded by a cold perfusion, abolished the granular material in the developing enamel, whereas the granular material in the extracellular spaces between Tomes' processes remained as huge droplets of dense material. These results indicate that at least a part of the matrix protein of rat incisor developing enamel has the physico-chemical property to dissociate at low temperature during pre-wash perfusion and move toward the enamel surface. Thus, in perfusion-fixed specimens, an intimate relationship between the artifactual formation of stippled material and the temperature of the pre-wash perfusate is suggested.

scholarly journals Ultrastructural demonstration of cytochrome oxidase via cytochrome C in cerebral cortex.

1979 ◽  
Vol 27 (9) ◽  
pp. 1261-1266 ◽  
Author(s):  
S Y Al-Ali ◽  
N Robinson
Keyword(s):  
Enzyme Activity ◽  
Cerebral Cortex ◽  
Cytochrome C ◽  
Cytochrome Oxidase ◽  
Staining Pattern ◽  
Oxidative Polymerization ◽  
Intermembrane Space ◽  
Vascular Perfusion ◽  
Cytochemical Localization ◽  
Perfusion Fixation

A procedure for the ultrastructural cytochemical localization of cytochrome oxidase via cytochrome c in the cerebral cortex is described. Vascular perfusion fixation by formaldehyde and glutaraldehyde of different concentrations and mixtures of the two gave varying results. A mixture of 4% formaldehyde and 0.5% glutaraldehyde gave the best combination of ultrastructural preservation and retention of enzyme activity. Histochemical methods were examined for optimum incubation conditions, based on the oxidative polymerization of 3,3'-diaminobenzidine (DAB) to an osmiophilic product. The reaction product was discretely localized within intercristate and the intermembrane space of mitochondria. The staining pattern was the same in nerve cells and in neuroglia and their processed. The DAB reaction product was also found in mitochondria of the endothelial cells.

Perfusion Fixation of Whole Fish for Electron Microscopy

1975 ◽  
Vol 32 (3) ◽  
pp. 416-422 ◽  
Author(s):  
D. E. Hinton
Keyword(s):  
Microscopic Examination ◽  
Electron Microscopic Examination ◽  
Complete Removal ◽  
Cell Ultrastructure ◽  
Whole Body ◽  
Electron Microscopic ◽  
Vascular Perfusion ◽  
Liver And Kidney ◽  
Excellent Preservation ◽  
Perfusion Fixation

A method for obtaining uniform fixation of whole bodies of fish for electron microscopic study is described. The procedure employs vascular perfusion of aldehyde fixatives through the proximal aorta of anesthetized fish. Light microscopic examination of semi-thin (0.5–1 μ) sections of Epon embedded material, stained with toluidine blue, showed near to complete removal of blood cells from vessels of gill, bone, skin, skeletal muscle, heart, exocrine pancreas, mesentery, glomerulus, and brain. Electron microscopic examination of liver and kidney, fixed by whole body perfusion, showed excellent preservation of cell ultrastructure.

Morphometric analysis of normal canine Leydig cells and their organelles in sexually mature beagles

1987 ◽  
Vol 45 ◽  
pp. 816-817
Author(s):  
G. J. Argentieri ◽  
G. E. Visscher ◽  
R. L. Robison
Keyword(s):  
Leydig Cells ◽  
Fixation Method ◽  
Morphometric Data ◽  
Electron Microscopic ◽  
Vascular Perfusion ◽  
Baseline Information ◽  
Perfusion Fixation ◽  
Control Adult ◽  
Induced Changes

It was our goal to obtain quantitative baseline information on canine Leydig cells and their organelles. Little quantitative baseline data on canine Leydig cells exists in the literature which gives a comprehensive light microscopic (LM) and transmission electron microscopic (TEM) overview. Information is available for various species which correlates testicular physiological and morphometric data. Morphometric data on canine Leydig cells and their organelles becomes important when evaluating naturally occurring and experimentally induced changes during research and development.Four control adult male beagles (14 to 20 months old) were used in this study. A reliable in vitro vascular perfusion fixation method for the testis was developed. Initial buffer rinsing of the vasculature was accomplished using a vasodilator and an anticoagulant. Primary fixation was with 1% paraformaldehyde, 1.5% glutaraldehyde in a 0.1 M cacodylate buffered solution, followed by a second, doubly concentrated aldehyde fixative solution.

scholarly journals Effect of high transcapillary pressures on capillary ultrastructure and permeability coefficients in dog lung

2001 ◽  
Vol 90 (2) ◽  
pp. 638-648 ◽  
Author(s):  
Michael B. Maron ◽  
Zhenxing Fu ◽  
Odile Mathieu-Costello ◽  
John B. West
Keyword(s):  
High Pressure ◽  
Pressure Group ◽  
Blood Gas ◽  
Vascular Perfusion ◽  
Gas Barrier ◽  
Lung Lobe ◽  
Lower Lung ◽  
Perfusion Fixation ◽  
Very High ◽  
Lung Lobes

To determine the correlation between ultrastructural and physiological changes in blood-gas barrier function in lungs transiently exposed to very high vascular pressures, we increased capillary transmural pressure (Ptm) of 6 canine isolated perfused left lower lung lobe preparations (high-pressure group) to 80.3 Torr for 3.8 min and then determined the capillary filtration ( K fc) and osmotic reflection (ςd) coefficients at a Ptm of 19.1 Torr in the ventilated lung lobes. This was followed by perfusion fixation of the lobes at a Ptm of 20.5 Torr for ultrastructural analysis. These data were compared with those obtained in six lobes in which Ptm was not transiently elevated before K fc, ςd, and ultrastructural evaluation. K fc was higher [0.249 ± 0.042 (SE) vs. 0.054 ± 0.009 g · min−1 · Torr−1 · 100 g−1; P < 0.01] and ςd was lower (0.52 ± 0.07 vs. 0.85 ± 0.08; P < 0.01) in the high-pressure group. In contrast, although endothelial and epithelial breaks were occasionally observed in some experiments, their incidence was not increased in the high-pressure group. These data suggest that the increased transvascular water and protein flux occurred through pathways of a size not resolvable by electron microscopy after vascular perfusion-fixation at a Ptm of 20.5 Torr.

scholarly journals Zinc-aldehyde fixation for light-microscopic immunocytochemistry of nervous tissues.

1983 ◽  
Vol 31 (12) ◽  
pp. 1435-1438 ◽  
Author(s):  
E Mugnaini ◽  
A L Dahl
Keyword(s):  
Neuronal Cell ◽  
Tissue Penetration ◽  
Vascular Perfusion ◽  
Axon Terminals ◽  
Matrix Stabilization ◽  
Neuronal Cell Bodies ◽  
Low Concentrations ◽  
High Concentrations ◽  
Perfusion Fixation ◽  
Pap Method

Two procedures are described for vascular perfusion of the nervous system with a zinc-aldehyde fixative. The procedures, simple and economical, combine the advantages of perfusion fixation with an aldehyde solution and matrix stabilization by a mordating agent, and improve the sensitivity of the peroxidase-antiperoxidase (PAP) method for the immunocytochemical localization of several antigens. Procedure A is intended for the light-microscopic immunostaining of cellular elements containing high concentrations of antigen. Penetration of the immunoreagents is adequate without the use of detergents. Procedure B is particularly advantageous for the light-microscopic immunostaining of cellular elements that contain low concentrations of antigen, and for high-resolution microphotography. With procedure B, the tissue penetration of immunoreagents is more limited than with procedure A; however, neuronal cell bodies and dendrites are more easily penetrated by the immunoreagents than are axons. Neuronal cell bodies and dendrites thus become clearly detectable in the light-microscope, even when they are surrounded by numerous immunoreactive axon terminals, and especially after the blockage of axoplasmic transport by the topical injection of colchicine.

A technique for in vivo vascular perfusion fixation of the sheep central nervous system

1998 ◽  
Vol 79 (2) ◽  
pp. 195-199 ◽  
Author(s):  
Stephen Santoreneos ◽  
Marcus A Stoodley ◽  
Nigel R Jones ◽  
Chris J Brown
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Vascular Perfusion ◽  
Perfusion Fixation

Ultrastructural localization of neuropeptides in the rat brain

1978 ◽  
Vol 36 (2) ◽  
pp. 612-613
Author(s):  
F.W. Van Leeuwen
Keyword(s):  
Freeze Substitution ◽  
Ultrastructural Localization ◽  
Serial Sections ◽  
Microscopical Level ◽  
Electron Microscopical Level ◽  
Enzyme Method ◽  
Perfusion Fixation ◽  
Electron Microscopical ◽  
Unlabeled Antibody ◽  
Peroxidase Conjugate

In order to obtain specific and optimal ultrastructural localization of vasopressin and oxytocin in the hypothalamo-neurohypophyseal system of the rat, 2 staining procedures and several tissue treatments were evaluated using neurohypophyseal tissue. It appeared from these studies that post-embedding staining with the unlabeled antibody enzyme method developed by Sternberger allows greater dilution of the first antibody (anti-vasopressin, 1:4800) than the indirect procedure (1:320) using a peroxidase conjugate as second antibody. Immersion fixation with 4% formalin during 24 hours gave better results (general ultrastructure, immunoreactivity) than those obtained by perfusion fixation with 2.5% glutaraldehyde-1% paraformaldehyde or freeze substitution.Since no reliable specificity tests were performed at the electron microscopical level, tests were developed for antibodies against both vasopressin and oxytocin. For anti-vasopressin plasma neural lobes of homozygous Brattleboro rats, that are lacking vasopressin by a genet- ical defect, were used. For antibodies against both hormones serial sections were used that were alternately incubated with the antibodies.

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