Showing papers in "Biotechnic & Histochemistry in 1979"
TL;DR: Within the pH range 2.5-6.5 tungstic acid (an isopolyacid) prevents the reduction of silver ions by ascorbic or hydroquinone more effectively than either gum acacia or other protective colloids.
Abstract: Within the pH range 2.5-6.5 tungstic acid (an isopolyacid) prevents the reduction of silver ions by ascorbic acid or hydroquinone more effectively than either gum acacia or other protective colloids. The colloid state of tungstic acid can be stabilized with nonionic detergents, especially with Triton X-100. For buffering the system a mixture of acetic acid and sodium acetate is optimal. Physical developers constituted on the basis of the observations are, in contrast to those commonly used in histology, light insensitive, and remain clear for about 30 min at mom temperature 2-5 times as long as the time required for development.
82 citations
TL;DR: The sections embedded in plastic, i.e. water soluble methacrylate (Polyscience's JB-4) and cut at 2 micrometers, were far superior to frozen sections and paraffin embedded sections both in tissue detail and in the localization of the histochemical reaction product.
Abstract: Histochemical staining for enzymes is usually performed on frozen sections. This report lists the longer incubation times required to demonstrate esterase, acid phosphatase, β-galactosidase, and cytochrome oxidase in plastic embedded and routine paraffin embedded tissues. The sections embedded in plastic, i.e. water soluble methacrylate (Polyscience's JB-4) and cut at 2 μm, were far superior to frozen Sections and paraffin embedded sections both in tissue detail and in the localization of the histochemical reaction product.
74 citations
Journal Article•
59 citations
TL;DR: A reduced silver technique using physical development to stain embryonic nervous tissue is described, which yields uniform, complete and reproducible staining of axons at all developmental stages of the nervous tissue and is easy to handle.
Abstract: A reduced silver technique using physical development to stain embryonic nervous tissue is described. Brains are fixed in Bodian's fixative. Paraffin sections are pretreated with 1% chromic acid or 5% formol. They are impregnated with 0.01% silver nitrate dissolved in 0.1 M boric acid/sodium tetraborate buffer of pH 8 or with silver proteinate. Finally they are developed in a special physical developer which contains 0.1% silver nitrate, 0.01-0.l% formol as developed agent, 25% sodium carbonate to buffer the solution at pH 10.3, 0.1% ammonium nitrate to prevent precipitation of silver hydroxide, and 5% tungstosilicic acid as a protective colloid. The development takes several minutes in this solution, thus the intensity of staining can be controlled easily. The method yields uniform, complete and reproducible staining of axons at all developmental stages of the nervous tissue and is easy to handle.
57 citations
TL;DR: Semithin sections of tissues fixed for electron microscopy can be stained directly after mounting, or by a wider range of stains once the polystyrene has been removed by organic solvents, and given acceptable results.
Abstract: Polystyrene embedments of histological specimens can be Obtained with a solution 1 : 4 polystyrene-toluene, 5% benzyl alcohol and 1% dibutyl phthalate, allowing the solvent to evaporate in polyethylene containers for 2-3 days at 58 C. The resulting blocks are easily cut into truncated pyramids, each containing a piece of tissue. which are then glued to a Plexiglas support Drying is completed at 80 C for 20 hr. The pyramids can then be sectioned to produce thick sections, with a steel knife or to produce semi- or ultrathin sections with a glass knife. A 10% paraldehyde solution is used to mount the light microscopy dons on a slide heated on a hot plate to 80 C; those can be treated with the same techniques used with paraffin sections. The results are of high quality. Semithin sections of tissues fired for electron microscopy can be stained directly after mounting, or by a wider range of stains once the polystyrene has been removed by organic solvents. In electron-microscopy, the ultrathin sections obtained...
43 citations
TL;DR: A rapid, reliable silver impregnation method for nervous tissue fixed in formol-saline, Bouin or Susa, which is very reliable and selective, making it suitable for general routine and research use.
Abstract: A rapid, reliable silver impregnation method is described for nervous tissue fixed in formol-saline, Bouin or Sum. Sections are impregnated for 10-15 minutes at room temperature or 37 C in a solution containing 0.5 g Protargol-S, 0.005-0.01 g allantoin, 1 ml of 1% Cu[NO2]2, 1 ml of 1% AgNO3. and 1-2 drops of 30% H2O2 in 100 ml distilled water. Thereafter the dons arc reduced in a hydroquinone-formalin solution. This is followed by gold toning and subsequent reduction and mounting. Alternatively. following the first reduction, the silver image can be intensified by placing sections in a silver-allantoin bath which is followed by reduction and mounting. This method is very reliable and selective, making it suitable for general routine and research use.
39 citations
TL;DR: A quick, easy, and reproducible staining method using stable reagents which are readily available commercially but which may also be prepared in the laboratory.
Abstract: The esterases used to identify monocytes are best demonstrated using alpha-naphthyl butyrate as substrate However, the reagents commonly used for this stain are time-consuming to prepare and are unstable This report describes a quick, easy, and reproducible staining method using stable reagents which are readily available commercially but which may also be prepared in the laboratory
39 citations
TL;DR: Combination of Karnovsky's cholinesterase staining with silver impregnation of axons (modified Bodian's technique) offers a new means of studying the relation between the pre- and postsynaptic elements in the frog neuromuscular junction.
Abstract: Combination of Karnovsky's cholinesterase staining with silver impregnation of axons (modified Bodian's technique) offers a new means of studying the relation between the pre- and postsynaptic elements in the frog neuromuscular junction. The method can be applied to whole muscles so that synapses of individual superficial muscle fibers which have previously been investigated by electrophysiological techniques can be identified after staining. In this way synaptic activity can be correlated with such synaptic features as number of axon branches, length of the occupied synaptic gutter, axonal sprouts, etc. The distinction between occupied and unoccupied parts of the synaptic gutters is useful when studying reinnervation, regression, or growth of a synapse.
38 citations
TL;DR: Pectinase used for cell separation prior to cytophotometry contains a DNase that is able to penetrate the cells of pine root tips and attack nuclear DNA, and it is suggested that heat denaturation of the DNase should also be effective and might be used in combination with the magnesium chelators.
Abstract: Pectinase used for cell separation prior to cytophotometry contains a DNase that is able to penetrate the cells of pine root tips and attack nuclear DNA. When pine root tips were exposed to 1% pectinase (pH 6.0), there was a decrease in nuclear DNA content at every sample point and a sharp drop between 16 and 20 hr. The effect of the DNase was eliminated by preparing the enzyme solution in 0.01 M sodium citrate or 0.001 M EDTA. It is suggested that heat denaturation of the DNase should also be effective and might be used in combination with the magnesium chelators.
29 citations
TL;DR: Differences in cutting speed and cutting cycle cadence critical to quality sectioning with different embedments are detailed and factors important in choosing the best embedment and most appropriate section thickness as well as choosing between dry or wet sectioning methods are also considered.
Abstract: Experiments undertaken to improve the ease and quality of preparing tissue for light microscopy have resulted in methods for routine production of high quality serial paraffin and glycol methacrylate sections in the 1 to 4 micrometer range using a standard rotary microtome. These methods involve sectioning with mechanically-broken Ralph-type knives, inspected for quality before mounting with double-stick tape on a holder-fluid trough in which the ribbon floats over an immersed slide and sections are mounted by lowering the fluid level in the trough. Specimen return stroke retraction, necessary for quality sectioning of plastic or with a fluid trough, is inexpensively provided by an attachable specimen holder that fits into the rotary microtome's chuck and, actuated by the microtome mechanisms, retracts the specimen during each return stroke. Descriptions of four instruments: a mechanical breaker for Ralph knives, a knife edge inspection device, an attachable specimen retractor for rotary microtomes, and a section collecting-mounting device as well as details of their use are included. Differences in cutting speed and cutting cycle cadence critical to quality sectioning with different embedments are detailed. Factors important in choosing the best embedment and most appropriate section thickness as well as choosing between dry or wet sectioning methods are also considered.
28 citations
TL;DR: Hydrophilic resin blocks with adequate hardness and elasticity for semithin sectioning are made by addition of divinylbenzene and methylmethacrylate to a commercial embedding kit, and fine resolution and low distortion achieved are compensating gains.
Abstract: Procedures for obtaining sections 1 micrometer thick on a conventional rotary microtome are described. Hydrophilic resin blocks with adequate hardness and elasticity for semithin sectioning are made by addition of divinylbenzene and methylmethacrylate to a commercial embedding kit. The blocks are pinched between two simple adapters and mounted in the specimen holder of a microtome. A glass knife of the Ralph type with an effective blade length of 25 mm is made from a glass slide and attached to a metal bar with paraffin. The low cost assembly is set in the steel knife holder of a conventional rotary microtome. Sections 1 micron in thickness can be cut from the resin embedded blocks. Staining with the usual staining solutions may be weak due to the thinness of the sections, but the fine resolution and low distortion achieved are compensating gains.
Journal Article•
TL;DR: A method is described for a rapid and systematic light microscopic documentation of Golgi impregnated neurons while they are being sectioned for electron microscopy.
Abstract: A method is described for a rapid and systematic light microscopic documentation of Golgi impregnated neurons while they are being sectioned for electron microscopy. A drawing under the light microscope of a Golgi impregnated neuron is made first; subsequently thin door of the tissue containing this neuron are cut in the same plane as for light microscopy. During thin sectioning the chuck containing the block is taken out of the ultramicrotome at regular intervals and placed in a special device under a light microscope. The neuron is photographed to record the stage of sectioning. Comparison of the micrographs indicates which put of the and its dendritic tree are contained in the thin sections. No semithin sections are used and therefore no material is lost for reconstruction.
TL;DR: A new modification of the Snodgrass-Dorsey (1963) albumin embedding method is described, which produces high quality finished slides in which the stained brain tissue is surrounded by a colorless albumin-gelatin matrix.
Abstract: A new modification of the Snodgrass-Dorsey (1963) albumin embedding method is described. Formalin fixed brains of various ages of rhesus monkey (Macaca mulatta) were sunk in 10% phosphate buffered formalin which contained 30% sucrose, and then embedded in a 3% gelatin, 30% egg albumin solution which had been centrifuged to ensure uniformity. The albumin-gelatin was hardened in formaldehyde fumes and blocks cut frozen at 10-40 micron. Sections thus prepared can be handled easily and mounted without damage to the tissue. Modifications of conventional cell and fiber stains produce high quality finished slides in which the stained brain tissue is surrounded by a colorless albumin-gelatin matrix.
TL;DR: Acidified 2,2-dimethoxypropane (DMP) was found to be the only successful means of preparing the sugarbeet root maggot larva, Tetanops myopaeformis (Röder) (Diptera:Otitidae), for the scanning electron microscope.
Abstract: Acidified 2,2-dimethoxypropane (DMP), used as an alternative to regular fixation and dehydration methods for insects, was found to be the only successful means of preparing the sugarbeet root maggot larva, Tetanops myopaeformis (Roder) (Diptera:Otitidae), for the scanning electron microscope. No morphological changes were evident when DMP treated sugarbeet root maggot adults were compared to fresh (unfixed) adults and glutaraldehyde-osmium tetroxide fixed adults. The method has been used with success on several gropus of insects. Acidified CMP is quickly hydrolyzed by water in tissue to acetone and methanol. DMP is advantageous in that it penetrates water impermeable cuticles rapidly and saves several steps and time in the fixation and dehydration process.
Journal Article•
TL;DR: The four analogs comprising basic fuchsin have been separated using thin layer chromatography (TLC) using a solution of 25% methanol, 10% ammonium hydroxide, and 65% distilled water.
Abstract: The four analogs comprising basic fuchsin have been separated using thin layer chromatography (TLC). Mixtures spotted on reverse phase TLC plates were developed with a solution of 25% methanol, 10% ammonium hydroxide, and 65% distilled water. The Rf values of the analogs were for pararosaniline, 0.54; rosaniline, 0.41; magenta II, 0.31; new fuchsin, 0.19.
TL;DR: Changes in the agarose lymphocyte migration technique are described which resulted in satisfactory differentiation of T and B lymphocytes and monocytes which have migrated as a monolayer for 1-3 days.
Abstract: This report describes alterations in the agarose lymphocyte migration technique which resulted in satisfactory differentiation of T and B lymphocytes and monocytes which have migrated as a monolayer for 1-3 days. The Wright-Giemsa staining used in the original method did not permit identification of individual migrating cell types. The most important modifications were changing from a plastic to a glass migration surface, and significantly reducing the overlying thickness of agarose which permitted a short fixation time and easy preparation of permanent slides ruined for nonspecific esterase. The esterase staining of monocyte cytoplasm was intense and diffuse. One or two small, discrete areas of cytoplasmic esterase activity were identified in the majority of T lymphocytes B lymphocytes showed either a trace or no evidence of esterase activity. The modified method should prove useful for the histochemical differentiation of migrating subpopulations of mononuclear cells.
TL;DR: Combined treatment with cycloheximide plus 8-hydroxyquinoline was superior to treatments with either chemical separately and contained nearly ten times more cells in prophase and metaphase that were suitable for chromosome counting.
Abstract: The actions of cycloheximide and 8-hydroxyquinoline on dividing cells of root meristems of Zea mays L. have been studied during the development of a new cytological technique for sugar cane (Saccharum) root tips. The determination of mitotic phase indices revealed that combined treatment with cycloheximide (70 ppm) plus 8-hydroxyquinoline (250 ppm) was superior to treatments with either chemical separately. After the combined treatment, the preparations contained nearly ten times more cells in prophase and metaphase that were suitable for chromosome counting than those given a single pretreatment with 8-hydroxyquinoline. This new pretreatment has been developed especially for chromosome studies in tropical grasses with a large number of small chromosomes. However, both chemicals are active in a wide range of plant species.
TL;DR: The number of cells needed to achieve a monolayer is significantly reduced compared to the usual culture flasks, but are abundant enough to assess ultrastructural changes accurately.
Abstract: Plastic coverslips made of polymethylpentene serve as excellent substrates for growth of bovine endothelial cells, and are easily processed for both transmission (TEM) and scanning (SEM) electron microscopy. Portions of the same coverslip (monolayer) are used for both SEM and TEM examination and are fixed, postfixed, and dehydrated as a single entity. The portion of the coverslip for SEM is then excised, critical point dried, and mounted for sputter coating prior to viewing. The remaining piece of coverslip used for TEM is Epon-Araldite embedded, polymerized, separated from the coverslip by liquid nitrogen immersion, and sectioned either "en face" or in cross section for viewing. Coated glass coverslips are not required and organic solvents such as propylene oxide, acetone, and amyl acetate can be used for dehydration and infiltration. Furthermore, specimens do not require re-embedding or blocks to be glued onto blank capsules before sectioning. The number of cells needed to achieve a monolayer is significantly reduced compared to the usual culture flasks, but are abundant enough to assess ultrastructural changes accurately. Support films may be required to prevent folding of the ultrathin section which can obstruct viewing of cells located on the edge of the section.
TL;DR: It was concluded that the vacuoles of the companion cells of the potato tuber were stained by the ion trap mechanism because of the color of the accumulated stain, the lack of staining when neutral red was applied in an acidic solution, and the complete destaining after soaking in dilute ammonium hydroxide.
Abstract: When staining the internal phloem region of a potato tuber with the vital stain neutral red, it was observed that files of elongated cells of narrow diameter were heavily stained and were easily distinguishable from the more isodiametric parenchyma cells, many of which did not stain with neutral red. The elongated cells were identified as companion cells by locating the adjacent sieve-tube members through counterstaining with aniline blue and reviewing under violet light. Of a number of other plants surveyed, only parsnip roots possessed companion cells exhibiting a similar slective staining. In other plants both the companion cells and the surrounding parenchyma cells usually stained. Sieve-tube members never accumulated neutral red. It was concluded that the vacuoles of the companion cells of the potato tuber were stained by the ion trap mechanism because of the color of the accumulated stain, the lack of staining when neutral red was applied in an acidic solution, and the complete destaining after soaking in dilute ammonium hydroxide.
TL;DR: Evaluation of the actual ratio of the observed TiCl2 dye content to the theoretical for pure zinc chloride methylene blue and comparison of spectroscopic and staining effects of graded hot dichromate oxid...
Abstract: Zinc chloride methylene blue appeared on the market almost contemporaneously with the zinc-free medicinal form. The former has rarely been reported as being used in blood stains. Recent suspension of manufacture of medicinal methylene blue by it. principal American producer has excited interest in the use of the zinc chloride form for the preparation of blood stains. According to Lillie (1944a,b) the azure B content of zinc chloride methylene blue may have varied from 5 to 30% in the samples studied. Taking the Merck Index (1968, 1976) figures for the spectroscopic absorption maximum (λmax) of 667.8 and 668 nm as standard, recent samples of zinc chloride methylene blue are calculated to contain 6-8% azure B. These figures are baaed on 1) the shift of λmax after exhaustive pH 9.5 chloroform extraction, 2) evaluation of the actual ratio of the observed TiCl2 dye content to the theoretical for pure zinc chloride methylene blue, 3) comparison of spectroscopic and staining effects of graded hot dichromate oxid...
TL;DR: A new RNA-specific fluorochrome, 4''-6-bis(2'-imidazolinyl-4'-5'H)-2-phenyl-4-phenoxyindole, visualizes the RNA-containing "partes fibrillares et granulares" of the nucleolus to analyze nucleolar structure and function by light microscopy.
Abstract: A technique for distinguishing nucleolar components by light microscopy is described. Silver staining is used for localizing nucleolar-organizing regions. A new RNA-specific fluorochrome, 4″-6-bis(2″-imidazoliny1-4′-5′H)-2-phenyl-4′-phenoxindole, visualizes the RNA-containing “partes-fibrillares et granulares” of the nucleolus. Both staining procedures can be combined to analyze nucleolar structure and function by light microscopy.
TL;DR: A method for the rapid and complete removal of methacrylic acid from commercial samples of glycol methacrylate is presented and sections of tissue embedded in polymer prepared from the purified monomer can be stained with basic dyes without simultaneously staining the polymer.
Abstract: A method for the rapid and complete removal of methacrylic acid from commercial samples of glycol methacrylate is presented. It entails conversion of the acid to an insoluble N-acylurea by treatment with an equivalent amount of N,N'-dicyclohexylcarbodiimide. Sections of tissue embedded in polymer prepared from the purified monomer can be stained with basic dyes without simultaneously staining the polymer.
Journal Article•
TL;DR: A method for the electron microscopic study of human peripheral lymphocytes by which very small buffy coats are obtained through centrifugation of heparinized whole blood in glass or plastic microhematocrit tubes is presented.
Abstract: A method for the electron microscopic study of human peripheral lymphocytes by which very small buffy coats are obtained through centrifugation of heparinized whole blood in glass or plastic microhematocrit tubes is presented. This method is time saving and efficient, yielding well preserved material and a comparatively large number of mononuclear cells (mainly lymphocytes) in each thin section.
TL;DR: A defendable procedure has been developed on an experimental basis and is reported here and Histochemical tests show that the functional group for myelin staining contains nitrogen, and probably hydrogen bonding is involved.
Abstract: Since Pearse in 1957 introduced chromoxane cyanine R as a dual nuclear and cytoplasmic stain there have appeared numerous procedures for use of this dye. These have differed widely, sharing in common mainly the implication that each is best. A defendable procedure has been developed on an experimental basis and is reported here. Four stock solutions are needed: (1) a 0.2% solution of chromoxane cyanine R in 0.5% aqueous H2SO4 (v/v); boil this solution for 5 min, (2) 10% FeCl3 in 3% HCl, (3) 1% aqueous HN4OH, and (4) 1% HCl in 70% ethanol. The staining solution: 40 ml of dye solution, 2 ml of FeCl3 solution, 8 ml H2O. Dewax and hydrate sections and stain for 10 min. If a myelin sheath stain is desired differentiate for 1 min in solution (3). For a nuclear stain differentiate for 1 min in solution (4). The nuclear stain when counterstained with eosin closely resembles the routine hematoxylin and eosin. Histochemical tests show that the functional group for myelin staining contains nitrogen, and probably hydrogen bonding is involved. The nuclear stain involves a different functional group and possibly neither electrostatic nor hydrogen bonding.
TL;DR: Polyhydroxybutyrate granules, generally poorly preserved in thin sections of Aquaspirillum serpens, were well preserved by the malachite green-glutaraldehyde fixative.
Abstract: Malachite green combined with glutaraldehyde has been used recently as a fixative for preserving and revealing lipid complexes in thin sections of eukaryotic cells examined by electron microscopy. When bacteria were prefixed with the above mixture granular electron dense inclusions were revealed in all cultures tested. These inclusions were replaced by electron transparent areas in cells fixed with glutaraldehyde alone. The structures were frequently located near to or within the nucleoid and adjacent to the cell membrane in Gram-negative bacteria and were associated with the nucleoid and mesosomes in Gram-positive species. Polyhydroxybutyrate granules, generally poorly preserved in thin sections of Aquaspirillum serpens, were well preserved by the malachite green-glutaraldehyde fixative. Malachite green complexes were observed outside of the cells in all preparations. Capsules were neither preserved nor stained.
TL;DR: A simple, inexpensive, reproducible method is described for prominently displaying the islets of Langerhans by sequential arterial perfusion of the organ of the sacrificed animal with saline, formalin, hematoxylin, and water, followed by clearing in methyl salicylate.
Abstract: A simple, inexpensive, reproducible method is described for prominently displaying the islets of Langerhans. The method consists of sequential arterial perfusion of the organ of the sacrificed animal with saline, formalin, hematoxylin, and water, followed by clearing in methyl salicylate. The procedure should be useful whenever islet tissue needs to be quantitatively distinguished from nonislet tissue and fixation is allowable.
TL;DR: A method for the cytophotometric estimation of ribonucleic acid in tissue sections using gallocyanin-chrome alum is described, and the results indicate that this method is of value in the quantitation of Ribon nucleic acid.
Abstract: A method for the cytophotometric estimation of ribonucleic acid in tissue sections using gallocyanin-chrome alum is described. The dye obeys Beer's law in gelatin sections. The effect of deoxyribonuclease on the staining of ribonucleic acid is also investigated. The results indicate that this method is of value in the quantitation of ribonucleic acid.