Characterization of isolated Fe-loaded rat hepatocytes prepared by collagenase perfusion

Summary Hepatocytes from livers of rats loaded by Fe-dextran treatment were isolated by an in situ collagenase perfusion technique and evaluated for their biochemical, cytochemical, and morphological characteristics in cell culture. Iron loads 15 times higher than in normal rat liver cells isolated in the same way were retained in the preparations with 40% present as hemosiderin. A simple centrifugation-mathematical approach is described for the calculation of Fe content in the hepatocyte (95%) and reticuloendothelial (5%) fractions in the isolates. The cells were cultured for 22 h without loss of protein synthesis capability or significant changes in cell count, viability, endogenous glutamate-oxaloacetate transaminase (GOT) or Fe and were morphologically similar in most respects to unloaded (normal) hepatocytes similarly cultured. Studies are in progress to assess the utility of these preparations as a model for Fe mobilization from Fe-loaded animals. This work is supported by National Institutes of Health Grants AM 25647-03 (M. Dawson, Principal Investigator) and GM 28158-01 (C. Tyson, Principal Investigator). The technical assistance of Mr. Jack E. Dabbs, Mr. Charles Hart, and Mr. Randy Douglas is acknowledged.     

Characterization of isolated Fe-loaded rat hepatocytes prepared by collagenase perfusion

Hepatocytes from livers of rats loaded by Fe-dextran treatment were isolated by an in situ collagenase perfusion technique and evaluated for their biochemical, cytochemical, and morphological characteristics in cell culture. Iron loads 15 times higher than in normal rat liver cells isolated in the same way were retained in the preparations with 40% present as hemosiderin. A simple centrifugation-mathematical approach is described for the calculation of Fe content in the hepatocyte (95%) and reticuloendothelial (5%) fractions in the isolates. The cells were cultured for 22 h without loss of protein synthesis capability or significant changes in cell count, viability, endogenous glutamate-oxaloacetate transaminase (GOT) or Fe and were morphologically similar in most respects to unloaded (normal) hepatocytes similarly cultured. Studies are in progress to assess the utility of these preparations as a model for Fe mobilization from Fe-loaded animals.     

Cinematography of Brain Structures Prepared by Perfusion Staining

Brains of rats were stained by perfusion, frozen, and sectioned. Of more than 10 stains tried only thionin and a silver stain (Weil-Davenport) gave good contrast upon penetrating the brain by way of its vascular system. Each section was discarded but the exposed surface of the block was photographed as single frames on 16 mm motion picture film. Proper projection at 16 frames/sec was obtained by cutting 10-14 μ sections, with 3 exposures after each section. Thus the viewer could readily visualize neural organization in 3 dimensions. The method is valuable for teaching neuroanatomy and for determining the extent of lesions in neurological investigations.     

Picrosirius red staining of dental structures

Histologic sections of dog mandibles and teeth were stained with picrosirius red and Mayer's hematoxylin. Collagenous structures of the mandible stained brilliant red. Dentinal tubules, Sharpey's fibers and other structures not easily seen in sections stained with hematoxylin and eosin alone were seen clearly after this procedure. Under polarized light collagen fibers could be specifically identified and their orientation determined. Picrosirius red-hematoxylin is recommended for examination of normal or pathologic dental specimens.     

Immunocytochemical staining of cytocentrifuge prepared cultured cells: nonspecific staining and its elimination

Summary In an attempt to localize hormones in cytocentrifuge-prepared cultured cells of small cell carcinoma of the lung (SCCL), various modifications of the immunoperoxidase (PAP) procedure (Sternberger, 1979) were tested. When using glutaraldehyde, formaldehyde, orp-benzoquinone fixation (Pearse & Polak, 1975) and rabbit antibodies in primary or bridging steps of the PAP procedure, nonspecific staining (false positives) could be elicited with the majority of rabbit antibodies tested, but not with antibodies from other animal sources. This problem could be eliminated by fixation of cells either with formalin-acetone (Masonet al., 1975) or, when using antibodies from a source other than rabbit, glutaraldehyde. It was not possible to localize ACTH in DMS-79, a human SCCL line known to produce this hormone. However, calcitonin was localized in the calcitonin-producing SCCL line DMS-53. Failure to localize ACTH in DMS-79 may be due to the lower levels of this hormone in DMS-79, as compared to the levels of calcitonin in DMS-53. This study emphasizes the importance of proper controls before concluding successful localization in a given immunocytochemical preparation of cultured cells.     

Selective staining of nucleic acid containing structures by uranyl acetate-lead citrate

The conventional uranyl acetate-lead citrate staining procedure has been modified to obtain selective staining of nucleic acid structures in glutaraldehyde fixed and epon embedded material. The selectivity of the stain is dependent on the concentration of uranyl acetate in the staining solution, the pH of the staining solution, the staining time and the washing conditions.     

Antikeratin antibody staining on ultrathin sections of epidermal cells prepared by low-denaturation embedding

Direct immunological identification of cellular components has not been possible in tissues prepared for electron microscopy by conventional methods. This may be attributed, in part, to the relatively harsh reagents employed. Using an approach to preparation of biological specimens for transmission electron microscopy that aims for minimal perturbation of native protein conformation, we have obtained specimens that may be stained with antibodies. Recent investigations using these methods have revealed new information regarding the organization of epidermal cells.     

Deterioration of limestone structures associated with copper staining

Sixty limestone monuments in Central London, UK, with copper staining from brass or bronze attachments, were surveyed. Severe spalling was noted at some copper-stained sites. The survey suggests that copper initially protects the surface, but causes stone deterioration after around 70 years. Samples from four limestone monuments were analysed microbiologically and using ESEM/EDX. EDX analyses suggested the build-up of salts below a basic copper carbonate skin (salting) as the most probable explanation of the observed deterioration. Copper-stained stone was free of photosynthetic microorganisms and yielded relatively few other microorganisms. ESEM and EDX analyses of a well-conserved limestone monument surface free of copper showed the almost complete conversion of surface limestone to gypsum. The grey/brown biofilms consisted of actinomycetes and filamentous fungi. The results indicate that copper initially prevents and then enhances limestone deterioration, and that atmospheric pollutants are far less harmful than phototrophic biofilms, since surface areas completely converted to gypsum are well preserved after more than 100 years.     

Highly purified mitochondria from rat brain prepared by phase partition

Mitochondria and synaptosomes from adult rat forebrain can easily be separated by counter-current distribution in an aqueous two phase system composed of Dextran T500 and poly(ethylene glycol) 4000. Both particles may also be separated by a batch procedure in which the same phase system is used. Electron micrographs and enzymatic activities show a high purity of the mitochondria obtained from the dextran-rich lower phase. Electron micrographs and enzymatic activities also show that intact synaptosomes can be obtained from the poly (ethylene glycol)-rich upper phase.The mitochondria purified by this method show good ADP/O ratios, respiratory control ratios, and state 3 rates. Synaptosomes showed a state 2-state 3 transition with no recuperation to state 4.     

Procedure for staining fixed human brain slices

The described method provides a new technique for differentiating areas of gray and white matter in fixed human brain slices. The technique is a modification of an existing method permitting use of the nonfading copper phthalocyanine dye alcian blue. Stained slices show turquoise gray matter that contrasts sharply with areas of white matter. Procedure: Cut human brains from gross anatomy laboratory cadavers into 4 mm slices and wash in running tap water for 12 hr. Oxidize slices in performic acid for 1.5 hr. Wash in running tap water for 12 hr. Stain slices in shallow dishes in 0.05% aqueous alcian blue. Wash in running tap water for 1 hr. Dry for 2-4 hr and embed in plastic.     

Uptake and release of norepinephrine by rat brain tissue fractions prepared by ultrafiltration

Abstract— Isolated nerve endings were removed from crude homogenates of rat brain by Millipore filters of pore size 0-5-0-8 μm. Synaptosomes containing L-[³H]norepinephrine were incompletely removed from a non-ionic medium by 0-8 μm pore filters, but were nearly quantitatively removed from Krebs’medium, as demonstrated by density gradient subcellular distribution. In suspension, synaptosomes accumulated labelled norepinephrine. Catecholamine uptake was active; it was inhibited by ouabain, imipramine, cocaine, β-phenethylamine and amantadine. Bound norepinephrine was released by a high concentration of K⁺. Nerve endings trapped on ultrafilters behaved very similarly to synaptosomes suspended in Krebs’medium by actively accumulating norepinephrine and serotonin; they also possessed monoamine oxidase activity and norepinephrine was released from them by increased concentrations of K⁺. Ultrafiltration provides a simple, rapid method of preparing metabolically active synaptosomes.