Microwaves Applied to Silver Impregnations with Ammoniacal Silver Carbonate

Techniques for impregnation with ammoniacal silver carbonate provide valuable information on all types of tissue; however, the time investment required to impregnate a few sections has limited their application. We have shortened the impregnation times by using microwaves in techniques for reticular fibers, astrocytes, nerve fibers and chromaffin cells. The results were satisfactory with markedly reduced impregnation time and elimination of nonspecific silver deposits.     

Problems and experience in the light optical and histological presentation of glia cells]

1. 8 histological techniques and 13 modifications derived from those were tested on usefulness for the demonstration of glial cells in the adult rat brain. From these methods the impregnation techniques of Golgi-Kopsch, Valenzuela y Chacón and Rio del Hortega were modified according to a scheme of variance to find out the optimal variants. 2. The impregnation quality depends on the animal species, the animal age, the health of brains, the brain area, the balanced proportion of the treatment stages and the biochemical state of the glial cells. 3. The silver impregnation techniques are not so specific that only one glial type is stained, but one type prevails. The silver carbonate procedure according to Hortega allows to impregnate oligodendrocytes, microglial cells and astrocytes in frozen as well as in paraffin sections. The method of Golgi-Kopsch is more suited for oligodendrocytes and microglial cells than for astrocytes. Following the procedure of Valenzuela y Chacón especially astrocytes, but also microglial cells allow impregnation in both frozen and paraffin sections. 4. The different demonstration qualities of the proved methods call for critical examination of absolute measurements of cell size, length of processes and ramification density. 5. The presence of cell groups of different disposition towards impregnation within a glial type speaks for a biochemical inhomogeneity of the glial types.     

Altered ultrastructure of cells of sunflower leaves having low water potentials

Summary Desiccation-induced alterations in cell structure were investigated in sunflower (Helianthus annum L.) leaves using light and electron microscopy. Desiccation was imposed by withholding water from the tissue, and all tissue fixation was carried out under isosmotic conditions. In addition to shrinkage of the vacuoles and intercellular spaces caused by water loss, the significant features of cell desiccation were the appearance of lipid droplets and vesicles close to dictyosomes, and plasmalemma and/or tonoplast breakage in the mesophyll cells. Breakage was followed by massive loss of cell organelles except for the thylakoid membranes of the chloroplasts, which retained much of their integrity even in the air-dried state. Plasmalemma and tonoplast disruption began in a few cells at water potentials of — 15 bars (relative water contents of 47%) and went to completion below —26 bars (relative water contents less than 28%) in the leaf mesophyll. Typically in this tissue, net photosynthesis becomes zero and the tissue becomes increasingly incapable of full rehydration at water potentials below — 20 bars. By contrast, water potentials of — 26 bars had no detectable effects on the phloem tissue. Structural alterations were little influenced by the rapidity of desiccation (a few minutes to as long as four days). It was concluded that desiccation-induced changes in cell structure are tissue-specific and occur on a cell-by-cell basis rather than in all cells of a tissue at once. The concentration of the cytoplasm and the disruption of the plasmalemma and/or tonoplast seem to be central events in the alteration of cell ultrastructure by desiccation.This research was supported by NSF grant GB41314.     

Modification of the Falck-Hillarp formaldehyde fluorescence method using the vibratome®: simple,rapid and sensitive localization of catecholamines in sections of unfixed or formalin fixed brain tissue

Summary Two modifications of the original Falck-Hillarp formaldehyde fluorescence technique are presented, both based on a recently introduced instrument, the Vibratome^®, which permits cutting of unembedded tissue with a section thickness down to 10 ,.The first modification involves sectioning of unfixed tissue at a temperature below +5°C, subsequent air drying and reaction with formaldehyde vapours. In the second procedure formalin fixed tissue is cut and processed as described above. It is essential that both formalin fixation and cutting of the fixed tissue takes place at a low temperature to avoid diffusion of the catecholamines.The results show that with both techniques central CA neurons can be visualized with a high degree of sensitivity. Furthermore, since the sections are free from fractures—a common problem in freeze-dried tissues—the Vibratome^® technique represents a valuable tool for mapping studies. It may also be added that since many steps of the original procedure are omitted the present techniques are also more rapid and simple. It is pointed out that using the Vibratome^® procedure on formalin fixed tissue, it will be possible to combine e.g. cholinesterase staining or Fink-Heimer silver impregnation or immunofluorescent studies with the Falck-Hillarp technique on serial or even on the same sections.     

Binding of I-Concanavalin a and agglutination of embryonic neural retina cells : Age-dependent and experimental changes

Embryonic chick neural retina cells dissociated from retina tissue by treatment with EGTA (a calcium chelator) show an age-dependent decline in ability to agglutinate with concanavalin A (ConA). This developmental change in cell surface properties is not due to loss of ConA-binding sites, since mature retina cells can be rendered agglutinable by mild trypsinization. It is also not due to masking of ConA receptors, or to a decrease in their amount, since retina cells from late embryos (19 days) bind four times as much ¹²⁵I-ConA as cells from early embryos (8 days). Our findings lead us to suggest that, as the retina differentiates the lateral mobility of ConA receptors in the cell membrane decreases resulting in a reduction of cell agglutinability; trypsinization of late embryo retina cells increases the mobility of the receptors and thereby facilitates their clustering by the lectin into a configuration conducive to cell agglutination.The ability of late embryo (19 day) retina cells dispersed with EGTA to agglutinate with ConA could be increased by still other treatments: by pre-incubation of the cell suspension in Tyrode's balanced salt solution (1 h, 37 °C); and by brief pre-exposure to glutaraldehyde. These two treatments did not enhance cell agglutination with wheat germ agglutinin (WGA). Glutaraldehyde treatment of trypsinized cells made them agglutinable with ConA also at 4 °C; cells treated otherwise agglutinated only at higher temperature. Surface-saturation of monodispersed retina cells with ConA at 37 °C—but not at 4 °C—prevented their agglutination with this lectin, but not with WGA; this inhibition was reversible by methyl a-D-glucopyranoside (αMG).     

Suppression of Connective Tissue Impregnation in a Silver Technique for Demonstrating Nerve Fibers

A tissue pretreatment technique is introduced which effectively suppresses the silver impregnation of connective tissue and nompecific background elements in peripheral nerve. The result is a selective impregnation of nerve fibers. The procedure utilizes fresh frozen sections and can be used with the Holmes (1947) or Bodian (1936) techniques. Fresh frozen sections are cut at 10 microns, mounted on slides and air dried for 5 minutes. They are fixed for 30 minutes in formol-sublimate (10% formalin saturated with mercuric chloride) and then placed into 0.5% iodine in 70% alcobol for 5 minutes followed by bleaching in 2.5% sodium thiosulfate for 2 minutes. After washing in running tap water for 10 minutes and a brief rinse in distilled water, impregnation is accomplished by the Holmes (1947) or Bodian (1936) procedure beginnins with the step containing the aqueous silver solution. The results show an absence of impregnation of connective tissue and nonspecific background. The technique is simple, rapid, and, by utilidng fresh hrozen sections, can be used for other histological and histochemical purposes. Several experiments were done to determine the causes of the connective tissue and background suppression. The air drying step was omitted; the sections were fixed in formalin without mercuric chloride; and the formol-sublimate fixation time was increased. The results suggest that connective tissue impregnation H suppressed by the use of mercuric chloride in the fixative and that the background supprgsion is related to the short fixation time with formol-sublimate.     

A rapid method combining Golgi and Nissl staining to study neuronal morphology and cytoarchitecture.

The Golgi silver impregnation technique gives detailed information on neuronal morphology of the few neurons it labels, whereas the majority remain unstained. In contrast, the Nissl staining technique allows for consistent labeling of the whole neuronal population but gives very limited information on neuronal morphology. Most studies characterizing neuronal cell types in the context of their distribution within the tissue slice tend to use the Golgi silver impregnation technique for neuronal morphology followed by deimpregnation as a prerequisite for showing that neuron's histological location by subsequent Nissl staining. Here, we describe a rapid method combining Golgi silver impregnation with cresyl violet staining that provides a useful and simple approach to combining cellular morphology with cytoarchitecture without the need for deimpregnating the tissue. Our method allowed us to identify neurons of the facial nucleus and the supratrigeminal nucleus, as well as assessing cellular distribution within layers of the dorsal cochlear nucleus. With this method, we also have been able to directly compare morphological characteristics of neuronal somata at the dorsal cochlear nucleus when labeled with cresyl violet with those obtained with the Golgi method, and we found that cresyl violet-labeled cell bodies appear smaller at high cellular densities. Our observation suggests that cresyl violet staining is inadequate to quantify differences in soma sizes.     

The correlation between uptake of Methyl Green and Feulgen staining intensity of cell nuclei. An image analysis study

Summary Paraffin sections of rat tissue fixed in either formaldehyde solution (3.6% w/v) or in Carnoy's fluid were stained using standardized Methyl Green—Pyronin procedures with the dyes used either simultaneously or in sequence. The sections were evaluated for the uptake of the two dyes by cell nuclei, nucleoli and cytoplasm using colour TV-image analysis. The parameters measured were integrated optical density and the surface area of the object. The sections were then destained and a Feulgen reaction was performed. The coordinates of the cells measured after the simultaneous Methyl Green—Pyronin method were stored in the computer, making it possible to measure the same cells in the Feulgen-restained sections. Image analysis gave results which invalidate the sequential methods as opposed to the simultaneous method. Mean optical densities were significantly increased for both dyes with the simultaneous method after formaldehyde fixation as compared to Carnoy fixation. The quantitative correlation of Methyl Green and DNA in the simultaneous technique was found to parallel exactly that of the Feulgen stain. In conclusion, the simultaneous Methyl Green—Pyronin technique is recommended while the sequential methods seem to be of less value.     

A comparison of microwaves and heat alone in the preparation of tissue for electron microscopy

Summary Microwaves have been used to stabilize tissues from the gastrointestinal tract for scanning electron microscopy. The temperature reached is important. Above 55–60°C, epithelial cell sheets begin to lift revealing the underlying basement membrane. These cells may be recovered from the supernatant by micropore filtration or the celloidin sock technique. At higher temperatures, produced either by microwave irradiation or in a water bath, more enterocytes are released. The epithelial cells are larger with increasing temperatures, less with microwaves than with heat alone or in the presence of formaldehyde. At 70°C and above, some proteins are lost and there is false localization of RNA. Some immunoperoxidase reactions are still positive after exposure of the tissue to 60°C. Tissues fixed in boiling formaldehyde retain a surprisingly good morphology.     

A method for silver impregnation of mammary myoepithelia

Histochemical methods for microscopic visualization of mammary myoepithelial cells all yielded considerable variation in completeness of myoepithelial cell staining. Although extremely variable, silver impregnation occasionally gave tissue sections containing myoepithelia having excellent microanatomical detail and contrast with other tissue elements. Consequently, sources of variation in the silver technique were considered. Composition of the tissue fixative and pH of the silver impregnating solution were most critical. A final method is presented which gives consistent, complete silver impregnation of myoepithelia, where both the cell body and cell processes are clearly evident. The staining procedure is not light sensitive, nor is acid cleaning of glassware necessary. Tissue sections from lactating mouse, rat, hamster and goat are presented; tissue from other species should stain as well. The procedure should greatly facilitate the study of the function of myoepithelial cells and the visualization of these cells in mammary pathology.     

A Method for Silver Impregnation of Mammary Myoepithelia

Histochemical methods for microscopic visualization of nummary myoepithelial cells all yielded considerable variation in completeness of myoepithelial cell staining. Although extremely variable, silver impregnation occasionally gave tissue sections containing myoepithelia having excellent microanatomical detail and contrast with other tissue elements. Consequently, sources of variation in the silver technique were considered. Composition of the tissue fixative and pH of the silver impregnating solution were most critical. A final method is presented which gives consistent, complete silver impregnation of myoepithelia, where both the cell body and cell processes are clearly evident. The staining procedure is not light sensitive, nor is acid cleaning of glassware necessary. Tissue sections from lactating mouse, rat, hamster and goat are presented; tissue from other species should stain as well. The procedure should greatly facilitate the study of the function of myoepithelial cells and the visualization of these cells in mammary pathology.     

Suppression of connective tissue impregnation in a silver technique for demonstrating nerve fibers.

A tissue pretreatment is introduced which effectively suppresses the silver impregnation of connective tissue and nonspecific background elements in peripheral nerve. The result is a selective impregnation of nerve fibers. The procedure utilizes fresh frozen sections and can be used with the Holmes (1947) or Bodian (1936) techniques. Fresh frozen sections are cut at 10 microns, mounted on slides and air dried for 5 minutes. They are fixed for 30 minutes in formol-sublimate (10% formalin saturated with mercuric chloride) and then placed into 0.5% iodine in 70% alcohol for 5 minutes followed by bleaching in 2.5% sodium thiosulfate for 2 minutes. After washing in running tap water for 10 minutes and a brief rinse in distilled water, impregnation is accomplished by the Holmes (1947) or Bodian (1936) procedure beginning with the step containing the aqueous silver solution. The results show an absence of impregnation of connective tissue and nonspecific background. The technique is simple, rapid, and, by utilizing fresh frozen sections, can be used for other histological and histochemical purposes. Several experiments were done to determine the causes of the connective tissue and background suppression. The air drying step was omitted; the sections were fixed in formalin without mercuric chloride; and the formol-sublimate fixation time was increased. The results suggest that connective tissue impregnation is suppressed by the use of mercuric chloride in the fixative and that the background suppression is related to the short fixation time with formolsublimate.     

A quantitative method for maceration of hydra tissue

Summary A method is described for the maceration (dissociation) of hydra tissue into single cells. The cells have characteristic morphology such that all basic types — epithelial, gland, mucous, interstitial, nematoblast, and nerve — can be distinguished. Criteria are given for identifying each cell type by phase contrast microscopy. It is shown that maceration quantitatively recovers cells from hydra tissue.     

Actin polymerization and synthesis in cultured neurones

The protein content of sympathetic neurones explanted from 10–11-day old chick embryos into culture medium containing nerve growth factor (NGF) increases steadily from about 100 to about 400 pg/cell in 7 days. Actin remains at close to 5% of the total protein during this period, but the proportion of unpolymerized actin falls. As measured by the inhibition of DNase I activity, rounded neurones without neurites contain 70 ± 7% of their total actin in monomeric form, whereas cells in mature, neurite-bearing cultures contain 39 ± 7%. When allowance is made for the increase in size of the neuronal cell bodies, the actin present in the neurites (‘axons’) alone is found to be almost entirely in filamentous form.Cultures exposed to radioactive leucine rapidly incorporate radioactivity into both sedimentable and non-sedimentable forms of actin. Actin-specific activities in the two fractions—estimated after isolation of the actin on small DNase I—Sepharose affinity columns—are similar after labelling for less than 1 h. Direct incorporation of newly-synthesized actin into filaments is suggested from these results.Pulse-chase experiments show that non-sedimentable protein in cultured sympathetic neurones turns over more rapidly than sedimentable protein. However, this is not true for actin, which shows a similar specific activity in sedimentable and non-sedimentable forms—even after 6 days of cold chase. This anomalous behaviour is simply explained by an exchange of actin molecules between filamentous and non-filamentous forms. Control experiments indicate that exchange does not occur to this degree during preparation of subcellular fractions. It is consequently attributed to exchange processes in the living cell.     

Morphological study of cell organelles during development. I - The nuclear sac and the endoplasmic reticulum on the rat nephron

The maturation of the endoplasmic reticulum (ER) of rat kidney tubule cells was studied with an osmium impregnation technique. Thick sections (0.3-0.6 micron) of kidney tissue were made after a five-day impregnation with osmium tetroxide and examined by standard transmission electron microscopy at 80-100 kV. Studies were performed on rat foetuses from 18-21 days of gestation, on newborns, and on 2-20 day old animals. At the undifferentiated stage, only a small percentage of the tubule cells were impregnated; in these, the perinuclear sac was stained and a few nuclear pores were already seen. Rudimentary, but thick canalicular projections seemed to originate from the perinuclear sac and become more extensive with maturity. Flattened saccules appeared later and fenestrations were seen in proximal tubule cells only when they seemed to have reached their functional specialization. In some cells, only the Golgi apparatus was stained. In the distal tubule cells, there was also progressive formation of a network consisting first of canaliculi and later of saccules which were rarely fenestrated. The osmium impregnation technique appears to be useful as an index of the ER organization development.     

Ultracytochemical evidence for the presence of GERL in pinealocytes of the Mongolian gerbil (Meriones unguiculatus)

Summary Ultracytochemical reactions for the demonstration of acid phosphatase, glucose-6-phosphatase and thiamine pyrophosphatase, as well as zinc iodide-osmium tetroxide impregnation, revealed the existence of GERL (Golgi apparatus — Endoplasmic Reticulum — Lysosomes) in pinealocytes of the Mongolian gerbil (Meriones unguiculatus). The spatial arrangement of this structure was studied on thick sections using a goniometric stage. Although it was not possible to determine whether GERL in pinealocytes belongs to the Golgi apparatus or to endoplasmic reticulum, it can be concluded that its presence in studied cells signifies that they are considerably more active synthetically than has been believed to date.     

Versatility of anti-horseradish peroxidase antibody-gold complexes for cytochemistry and in situ hybridization: preparation and application of soluble complexes with streptavidin-peroxidase conjugates and biotinylated antibodies

Summary In previous studies we have employed a gold-labelled, affinity-purified polyclonal antibody against horseradish peroxidase (anti-HRP — gold) in the avidinbiotin peroxidase complex (ABC) technique and indirect labelled avidin-biotin methods. The gold-labelled antibody was used as final revealing reagent to replace the 3,3-diaminobenzidine (DAB) reaction by immunogold silver staining. The anti-HRP — gold reagent proved to be advantageous since blocking of endogenous peroxidase activity in the tissue sections was not further required and staining of superior contrast and resolution could be achieved in paraffin sections. In the present study we have optimized this technique by combining the last two incubation steps, i.e. HRP-conjugated streptavidin and anti-HRP — gold. Different ratios of the two reagents were tested empirically to establish the conditions for the formation of a soluble complex with optimal staining properties. Quantitative evaluation by densitometry of the staining intensity showed that the soluble streptavidin-HRP/anti-HRP — gold complex and the indirect labelled avidin-biotin method employing the gold-labelled anti-HRP antibody performed equally well. Thus, the availability of this complex simplifies the streptavidin-biotin immunogold technique for immunohistochemistry, lectin histochemistry and in situ hybridization and further demonstrates the versatility of anti-HRP — gold complexes.     

Immunocytochemical detection of ricin. II. Further studies using the immunoperoxidase method

Summary Radio-iodinated ricin was injected into rat musclein vivo to establish the distribution of the toxin at various time intervals after injection. Injection site muscle and para-aortic lymph nodes were selected for localization of ricin by the immunoperoxidase technique. Sections of snap-frozen tissues were fixed using a variety of methods to establish the best protocol for the immunodetection method. This was found to be with an ether—ethanol mixture. Ricin was detected in tissue at the site of injection taken from rats sacrificed 1, 4, 8 and 24 h after injection and in tissue from animals dying from ricin intoxication after about 30 h. This method, however, failed to demonstrate unequivocally the presence of ricin in lymphoid tissue which had been indicated by the radiotracer study. The significance of these findings and their relevance to forensic diagnosis are discussed.     

Protein crystalloids in the stroma of bean plastids

Summary The formation of protein crystalloids in the stroma of bean plastids has been studied. It has been shown that, besides crystallization of the protein already present induced by water loss (which cannot be inhibited by chloramphenicol), the crystalloids also appear when the leaves are fed — but only in light — with very low concentrations of sucrose or glucose for one or two days. In this case their appearance is completely inhibited by simultaneous treatment of the leaves with chloramphenicol but not with cycloheximide. The process of crystalloid formation and disappearance has also been studied. The possibility of their removal from the plastids into the cell vacuoles has been followed and discussed.     

Amino acid auxotrophs from protoplast cultures of Nicotiana plumbaginifolia, Viviani

Summary Several amino acid requiring auxotrophs have been isolated from unsupplemented protoplast cultures of haploid Nicotiana plumbaginifolia following incubation with BUdR (1-5x10^-5, 2 days) and recovery on complete medium. The auxotrophic lines required the following amino acid(s) for growth: his, ile, leu, ile+val, met or try. Met^– is a new type isolated in higher plants. The same absolute amino acid requirement was observed in plants regenerated from auxotrophic cultures. Precursor feeding tests, enzyme assays, and/or metabolic complementation through protoplast fusion were used to identify the genetic lesion leading to auxotrophy. Mutant seeds were obtained from supplemented Met^– plants. Seeds were also collected from selfed plants regenerated from various complementing fusion products, and a His^– revertant. Genetic analysis indicated that under natural conditions of seed formation amino acid auxotrophy-in contrast to NR deficiency-failed to segregate in progeny tests.Abbreviations and definitions BUdR and FUdR 5-bromo- and fluoro-deoxyuridine respectively - AP imidazole acetol phosphate - IGP imidazole glycerol phosphate - NR nitrate reductase - NAA naphthaleneacetic acid - BAP 6-benzylaminopurine - TIP total isolation procedure - ER Escape rate—the proportion of the selected cell population surviving the BUdR treatment - BR Recovery rate—the proportion of clones identified as amino acid auxotrophs from total escaping clones - TS Total surviving colonies—the number of inoculated protoplasts/variant x plating efficiency - TST Total starvation time—the number of days on minimal medium (preincubation time+BUdR incubation time). The relationship days vs. number of divisions is as follows: 3- to 4-day-old protoplasts, 1 division; 5–6 days, 2 divisions; 7–8 days, 3 divisions Dedicated to Professor Georg Melchers to celebrate his 50-year association with the journal