A new fixative which primarily stabilizes carbohydrate moieties was developed for immunoelectron microscopy. It contains periodate, lysine and paraformaldehyde. Theoretically, the carbohydrates are oxidized by periodate and cross-linked by lysine. The fixative can preserve antigenicity as well as paraformaldehyde and ultrastructure as well as glutaraldehyde. Using this fixative and the peroxidase-labeled antibody technique, basement membrane antigen was localized within the cisternae of endoplasmic reticulum of parietal yolk sac cells and in extracellular basement membranes with adequate tissue successfully accomplished by conventional

Periodate-lysine-paraformaldehyde fixative a new fixative for immunoelectron microscopy

Research in autophagy continues to accelerate,(1) and as a result many new scientists are entering the field Accordingly, it is important to establish a standard set of criteria for monitoring macroautophagy in different organisms Recent reviews have described the range of assays that have been used for this purpose(2,3) There are many useful and convenient methods that can be used to monitor macroautophagy in yeast, but relatively few in other model systems, and there is much confusion regarding acceptable methods to measure macroautophagy in higher eukaryotes A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers of autophagosomes versus those that measure flux through the autophagy pathway; thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from fully functional autophagy that includes delivery to, and degradation within, lysosomes (in most higher eukaryotes) or the vacuole (in plants and fungi) Here, we present a set of guidelines for the selection and interpretation of the methods that can be used by investigators who are attempting to examine macroautophagy and related processes, as well as by reviewers who need to provide realistic and reasonable critiques of papers that investigate these processes This set of guidelines is not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to verify an autophagic response

/pdf/guidelines-for-the-use-and-interpretation-of-assays-for-4sx3t3he7d.pdf

Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes

Water is the most abundant component of biological material, but it is systematically excluded from conventional electron microscopy. This is because water evaporates rapidly under the vacuum conditions of an electron microscope. Cryoelectron microscopy has long been seen as a possible avenue to overcome this limitation, but until recently the direct observation of frozen-hydrated specimens was relatively unsuccessful because of a number of serious difficulties. These were, in particular, due to the absence of a good cryospecimen holder, the inherently low contrast of hydrated specimens and the structural damage due to ice crystals formed during freezing. As a consequence, the cryomethods which have flourished in electron microscopy during the last 20 years were not aimed at preserving the hydration of the specimen in the electron microscope. Freezing was only used as an aid to preparation. The objects ultimately observed in the electron microscope were dry and at room temperature. Such cryomethods have recently been reviewed in detail (Robards and Sleytr 1985).

/pdf/cryo-electron-microscopy-of-vitrified-specimens-46t0snibk6.pdf

Cryo-electron microscopy of vitrified specimens.

To determine the mechanism of the cardiac dilatation and reduced contractility of obese Zucker Diabetic Fatty rats, myocardial triacylglycerol (TG) was assayed chemically and morphologically. TG was high because of underexpression of fatty acid oxidative enzymes and their transcription factor, peroxisome proliferator-activated receptor-α. Levels of ceramide, a mediator of apoptosis, were 2–3 times those of controls and inducible nitric oxide synthase levels were 4 times greater than normal. Myocardial DNA laddering, an index of apoptosis, reached 20 times the normal level. Troglitazone therapy lowered myocardial TG and ceramide and completely prevented DNA laddering and loss of cardiac function. In this paper, we conclude that cardiac dysfunction in obesity is caused by lipoapoptosis and is prevented by reducing cardiac lipids.

https://www.pnas.org/content/pnas/97/4/1784.full.pdf

Lipotoxic heart disease in obese rats: implications for human obesity.

When biochemical studies on the liver are interpreted, the cells of the sinusoidal area frequently receive little attention because, compared to hepatocytes, their contribution to subcellular fractions is assumed insignificant. A systematic stereological analysis of liver parenchyma was therefore performed in order to determine the distribution of organelles and membranes between hepatocytic and nonhepatocytic cells, namely endothelial, Kupffer, and fat-storing cells. The livers were fixed by vascular perfusion and the data were corrected for systematic errors dur to section thickness and compression. The extracellular space compartment includes the lumina of sinusoids (10.6%), the space of Disse (4.9%), and the bile canaliculi (0.4%). Hepatocytes constitute 78% of parenchymal volume; the nonhepatocytes account for 6.3% and consist of 2.8% endothelial cells, 2.1% Kupffer cells, and 1.4% fat-storing cells. The nonhepatocytes contribute 55% of the volume of lipid droplets in the liver, 43% of the lysosomes, and 1.2% of the mitochondria. Although the nonhepatocytes account for only 8% of the total surface area of parenchymal membranes, they contain 26.5% of all the plasma membranes, 32.4% of the lysosomal membranes, 15.1% of the Golgi apparatus 6.4% of the endoplasmic reticulum, and 2.4% of the mitochondrial membranes. The data demonstrate the extent to which nonhepatocytic organelles can potentially contaminate subcellular fractions used for biochemical studies. Particularly important for the interpretation of studies on lysosomes, plasma membrane, and Golgi apparatus is the finding that an appreciable part of these organelles may be derived from cell types other than hepatocytes.

/pdf/distribution-of-organelles-and-membranes-between-hepatocytes-1x84egjs0o.pdf

Distribution of organelles and membranes between hepatocytes and nonhepatocytes in the rat liver parenchyma. A stereological study.

principles and techniques of electron microscopy principles and techniques of electron microscopy electron microscopy and optics: general textbooks principles and techniques of electron microscopy principles and techniques of electron microscopy principles and techniques of digital holographic microscopy scanning electron microscopy cathodoluminescence of quartz introduction to transmission/scanning transmission principles and technique of fluorescence microscopy by m principles and techniques of electron microscopy: volume 1 principles of the phase contrast (electron) microscopy principles and techniques of electron microscopy lecture 1 the principles of microscopy principles in light microscopy scanning techniques in electron microscopy

Principles and Techniques of Electron Microscopy

15. Beckman DSA-560.) Abell, L. L., Levy, 8. B., Brodie. B. B., and Kendall, F. E. Cholesterol in Serum, pp. 26-33, in Standard Methods of Clinical Chemistry, Vol. 11, D. Seligson (Ed. in Chief). Academic Press, Inc., New York, 1958. (Adapted to Beckman DSA-560,) American Optical Refractometer, American Optical Corp., Buffalo, NY. Trayser, K. A., and Seligson, D. A new "kinetic" method for enzyme analysis suitable for automation. Clin. Chem. 7 5 : 452,1969. (Adapted to Beckman DSA-560.) Bowers, G. N., and McComb, R. B. Continuous spectrophotometric method for measuring the activity of serum alkaline phosphatase. Clin, Chem. 22: 70,1966. (Adapted to Beckman DSA-560.) Poutsiaka, J. W., Keysser, C. H., Thomas, B. G. H., and Linegar, C. R. Simultaneous determination in dogs of liver a n d k idney functions with bromosulfalein and phenolsulfonephthalein. Toxicol. Appl. Pharrnacol. 4 : 55, 7 962. (Plasma BSP concentration determined on sample obtained 30 min after intravenous injection of 0.2ml of 89.5 pM BSP per kg body weight.) Instrumentation Laboratory Flame Photometer, Instrumentation Laboratory, lnc., Uxbridge, MA.

https://journals.sagepub.com/doi/pdf/10.1177/019262337400200306

Advanced Techniques in Biological Electron Microscopy

Administration of diethylnitrosamine I.P. at dose level of 2 mg/kg five days a week to young Sprague-Dawley rats induced foci of altered hepatocytes entirely devoid of glucose-6-phosphatase and adenosine triphosphatase activity. Several weeks later, these foci developed hepatocellular carcinoma mostly of the trabecular type. Although these "preneoplastic" altered cells appeared as normal hepatocytes when examined in HE-sections, they were found to exhibit sites of dedifferentiation with ultrastructural features of hepatoma cells. Early morphological changes indicative of neoplastic transformation took place first as excessive storage of glycogen then as marked alterations at the surface membranes of such cells. Striking increase in coated pinocytotic vesicles appeared along the apposing cell membranes associated with loss of sinusoidal microvilli, protrusion of cytoplasmic blebs in the intercellular spaces, reduction or loss of desmosomes and obliteration of bile canaliculi. Subsequent changes were seen as dilation and disruption of the rough endoplasmic reticulum (RER), dispersal of ribosomes, proliferation and clustering of the smooth endoplasmic reticulum (SER), predominance of annulate lamellae, depletion of glycogen, enlargement of the Golgi cisterns and segregation of nucleolar elements. After 20 weeks, rats given DENA at a dose level of 25 mg/kg twice a week developed pronounced cirrhotic nodules in addition to multiple hepatocellular carcinoma. In contrast to the neoplastic nodules, the cirrhotic ones retained normal activity of glucose-6-phosphatase and adenosine triphosphatase and exhibited ultrastructural features typical of normal hepatocytes.

https://journals.sagepub.com/doi/pdf/10.1177/019262338201000208

F. George Zaki

Papers

Principles and Techniques of Electron Microscopy

Advanced Techniques in Biological Electron Microscopy

Ultrastructure of altered hepatocytes induced by diethylnitrosamine (DENA).

International Symposium on Hepatotoxicity

Regulation of Hepatic Metabolism