Simple Methods for Mammalian Chromosomes

The ordinary Feulgen-squash technic, after acetic-alcohol fixation, provides a simple and reliable method of preparing many mammalian tissues for chromosome counts. Tissues best suited to the technic were the seminiferous tubules. Small pieces of tissue about 3-6 mm. long and 1-2 mm. wide were immersed in a freshly made fixative (composed of 3 parts absolute ethyl alcohol and 1 part glacial acetic acid) immediately after removal by biopsy or from a freshly killed animal. After fixation for 3-12 hours the tissue was stained by the standard Feulgen procedure after hydrolysis for 12 minutes in normal HCl at 60 °C A 1-2 mm. piece was then teased apart on a slide in 45% acetic acid with a pair of mounted needles, and the teased tissue was squashed between the slide and the cover slip. During squashing the pressure was applied by hand and was regulated so as to avoid any excessive scattering of the chromosomes. The preparations were made permanent by dehydrating and mounting in Euparal.     

Orseillin Bb for Staining Fungal Elements in Sartory's Fluid

A method is described for the squashing and permanent mounting of preparations, similar to the nucleal squash technic, to be used after hematoxylin, coal tar dyes and other stains. The colored pieces are immersed in glycerin, squashed or smeared, the glycerin is slowly removed with water by capillarity and the water is afterwards similarly substituted by alcohol. The cover glass is then removed and after a rapid rinsing in alcohol the materials are permanently mounted in alcohol-soluble resin.     

A Simple Method For Squashing And Mounting Preparations After Any Stain

A method is described for the squashing and permanent mounting of preparations, similar to the nucleal squash technic, to be used after hematoxylin, coal tar dyes and other stains. The colored pieces are immersed in glycerin, squashed or smeared, the glycerin is slowly removed with water by capillarity and the water is afterwards similarly substituted by alcohol. The cover glass is then removed and after a rapid rinsing in alcohol the materials are permanently mounted in alcohol-soluble resin.     

Elevated plasma testosterone concentrations during stallion-like sexual behavior in mares (Equus caballus)

Mounting interactions in mares isolated from stallions and the relationship to stage of the estrous cycle and level of circulating hormones were studied for 3 years in a herd averaging 105 mares. Mares were assigned to mounting, standing, and control groups. A control mare was selected by being within 1 day of the number of days after ovulation in a mounting mare. A total of 15 mounting interactions were detected by chance observation during the 3 years. A blood sample was collected immediately after the mounting interaction from each mare in the three groups, and a transrectal ultrasonographic examination of the reproductive tract was done. Two mounting interactions occurred during the early luteal phase and 13 during the follicular phase. The interactions that occurred during the follicular phase were used for comparisons among groups. The interval between mounting and the next ovulation, diameter of the two largest follicles, and the number of follicles larger and smaller than 20 mm were not different significantly among the mounting, standing, and control groups. Testosterone concentrations were higher (P<0.01) in the mounting group (17.7+/-2.3 pg/ml) than in standing group (10.9+/-0.5 pg/ml), and the difference between the mounting group and the control group (12.8+/-0.6 pg/ml) approached significance (P<0.08). Concentrations of androstenedione, estradiol, estrone, and progesterone did not differ significantly among groups. Results indicated that mounting behavior between mares is rare, usually occurs during the follicular phase, and is related to high circulating concentrations of testosterone.     

Sexual dimorphism in the motor mounting pattern of the New Zealand white rabbit: steroid regulation of vigor and rhythmicity of pelvic thrusting

The motor mounting patterns of male and female New Zealand white rabbits were analyzed by means of an accelerometric technique and frequency analysis. Clear behavioral dimorphism was noted in the motor mounting pattern. Pelvic thrusting by males was periodic while that performed by females lacked rhythmicity. Thrusting in males was more vigorous than in females. Ovariectomy markedly decreased the incidence of mounting behavior. Testosterone propionate (TP, 5 mg daily for 1 month), restored mounting in all ovariectomized rabbits. TP stimulated the vigor of thrusting and induced a rhythmic mounting pattern in many cases similar to that displayed by intact male rabbits, i.e., thrusting frequency 13-16 per second. Estradiol benzoate (10 micrograms daily for 1 month), elicited mounting in three of the seven rabbits tested. Pelvic thrusting in these rabbits was often highly synchronous showing a frequency higher (18 to 21 thrusts per second) than that displayed by male rabbits. The results suggest the following conclusions: (a) the behavioral dimorphism in mounting observed in rabbits is due to variations in the secretion of sex steroids by the adult gonads rather than to differences in the organization of the neural substrate of mounting; (b) gonadal steroids influence directly or indirectly the neural structures involved in some characteristics of pelvic thrusting, i.e., rhythmicity and vigor.     

Egg albumin embedding: A procedure compatible with neurological staining techniques

A water-soluble embedding medium for frozen sections of central nervous system and peripheral nerves which will tolerate on-the-slide alcohol dehydration, xylene clearing and synthetic resin mounting can be prepared by dissolving 20 gm of powdered egg albumin in 30 ml of distilled water. Formalin-fixed, thoroughly washed tissue is immersed in the albumin solution for several hours and then embedded in it. For embedding, a two-layered box is constructed with an inner layer of vegetable parchment paper. This is supported by an outer layer of aluminum foil with 6-8 perforations about 0.5 nun in diameter in its bottom. Hardening of the tissue-albumin block is achieved by formalin dialysis through the parchment paper when the box is stood for 24-48 hr in a layer of 25% formalin that wets only the bottom of the box. Frozen sections from such blocks are compatible with Nauta silver impregnations for degenerating axons, Cajal's gold chloride-sublimate for astrocytes and Pen-field's modified silver carbonate for oligodendroglia and microglia. These, as well as Nissl and myelin sheath preparations show minimal shrinkage and distortion.     

Muscarinic acetylcholine receptors in rat gastric mucosa. A radioautographic study using a potent muscarinic antagonist, 3H-pirenzepine

The muscarinic cholinergic innervation of the rat gastric mucosa was investigated by localizing the muscarinic receptors using a tritiated muscarinic antagonist, pirenzepine. Radioautography was performed by freeze drying stomach tissue, which was then embedded in Epon and wet sectioned with ethylene glycol, and dry mounting on emulsion film by the wire-loop method to prevent loss of the labelled substance during fixation and the radioautographic procedure. Light and electron microscopy showed that the specific pirenzepine-binding sites were localized predominantly on parietal cells, chief cells and perivascular plexuses. Analysis of the grain distribution on parietal cells revealed that the silver grains corresponding to the pirenzepine-binding sites were mainly on the basolateral plasma membrane. On the other hand, the surface mucous or mucous neck cells had few pirenzepine-binding sites.     

Muscarinic acetylcholine receptors in rat gastric mucosa

Summary The muscarinic cholinergic innervation of the rat gastric mucosa was investigated by localizing the muscarinic receptors using a tritiated muscarinic antagonist, pirenzepine. Radioautography was performed by freeze drying stomach tissue, which was then embedded in Epon and wet sectioned with ethylene glycol, and dry mounting on emulsion film by the wire-loop method to prevent loss of the labelled substance during fixation and the radioautographic procedure. Light and electron microscopy showed that the specific pirenzepine-binding sites were localized predominantly on parietal cells, chief cells and perivascular plexuses. Analysis of the grain distribution on parietal cells revealed that the silver grains corresponding to the pirenzepine-binding sites were mainly on the basolateral plasma membrane. On the other hand, the surface mucous or mucous neck cells had few pirenzepine-binding sites.     

Physiological adaptations to head-out aquatic exercises with different levels of body immersion

The purpose of this study was to compare the physiological adaptations to basic head-out aquatic exercises with different levels of body immersion. Sixteen young and clinically healthy subjects (9 women and 7 men) volunteered to participate in this study. Each subject performed 3 repetitions (on land, immersed to the hip, and immersed to the breast) of the aquatic exercise "rocking horse" for 6 minutes. The rating of perceived effort (RPE), the maximal heart rate achieved during the exercitation (HRmax), the percentage of the maximal theoretical heart rate estimated (%HRmax), the peak of oxygen uptake during the exercise (V(.-)O2peak), and the energy expenditure (EE) were evaluated. The RPE was significantly higher when exercising immersed to the hip than on land (p < 0.01) and immersed to the breast (p = 0.03). The HRmax and %HRmax were significantly lower when exercising with immersion to the breast than on land (p < 0.01) and with immersion to the hip (p < 0.01). The V(.-)O2peak was significantly different between all conditions. The lower mean value was verified when exercising immersed to the breast, followed by immersion to the hip and on land. The EE was significantly higher when performing aquatic exercises on land than when immersed to the hip (p = 0.02) and the breast (p < 0.01). So, physiological responses when exercising immersed to the hip are higher than when immersed to the breast. The physiological responses when exercising on land are higher than when exercising with immersion to the hip and to the breast.     

Does intermale mounting function as a dominance demonstration in rhesus monkeys?

The relationship between dominance and intermale mounting was analyzed in two troops of captive rhesus monkeys (Macaca mulatta). The data did not support the assumption that mounting among males functions as a dominance demonstration as described in the literature because mounting commonly (63% of 65 dyads) occurred in a bidirectional fashion, and mounting not only was performed by dominant males but also was shown with regularity (36% of 521 cases) by subordinate males. Intermale mounting was often (37% of 521 cases) associated with amicable interactions--commonly preceding them--but it was only rarely (2%) associated with dominance-subordination behaviors. It was concluded that intermale mounting may serve as a socially cohesive behavior in rhesus monkeys by promoting nonagonistic contact.     

Induction of male sex behavior in ewes with silastic implants containing testosterone propionate

In the first of two experiments, ovariectomized ewes with silastic implants containing testosterone propionate had higher concentrations of plasma testosterone than ovariectomized ewes with silastic implants containing testosterone (4.0 +/- .2 vs .8 +/- .1 ng/ml). In the second experiment, one or two 10 cm silastic implants filled with testoster-one propionate increased concentrations of plasma testosterone and induced male sex behavior in ovariectomized ewes similar to plasma testosterone levels and male sex behavior in rams. The interval from initiation of the treatment to the first behavior test in which mounting was consistently observed was 14.4 +/- 2.9 days and once mounting behavior was consistently induced, the testosterone treated ewes mounted estrous ewes an average of 10.7 +/- 1.3 times during a 10-minute test.     

Photopolymerized 2-Hydroxyethylmethacrylate as a mounting medium preserving immunocytochemical reaction and nuclear counterstain

Immunocytochemical or cytoenzymological techniques often make use of coupling reactions between a substituted naphtol and a diazonium salt. A positive reaction results in an azo dye precipitate. Unfortunately, this precipitate is easily soluble in alcohols and organic solvents. Thus, usual mounting media are not usable and permanent preparations cannot be obtained. A stable mounting medium such as Apathy's syrup can be used, but nuclear counter-staining disappears in a few days. We tested the hydrophilic monomer 2-hydroxyethylmethacrylate, containing various photopolymerizing agents, as a permanent mounting medium. 2-2 Dimethoxy 2-phenyl acetophenone proved to be the most useful photopolymerizer. The cytocentrifugation slides must be dried before mounting to avoid recrystallization artifacts. The azo dye precipitate and nuclear counterstaining can be preserved perfectly long-term. The cost of these media is very low.     

The use of basic Fuchsin in Plant Anatomy

A method useful in tracing the vascular bundles of herbaceous plants and the water conducting system of woody plants is presented. A stock solution of 2.5% basic fuchsin in 95% alcohol is prepared. A mixture of 1 to 2 parts of the stock solution to 100 parts of tap water (slightly alkaline) is made, standing 12-48 hours and being filtered before use. Cut ends of the living plants to be examined are immersed in the working solution until the dye appears in the parts desired.

The material may be examined by serial, hand sections mounted in 50% to 75% glycerin, or it may be split, laid open, and examined with a low power lens. Clearer views may be procured by dehydrating in an alcohol series and clearing in methyl salicylate. Permanent mounts may be made by removing the methyl salicylate with xylol and mounting in balsam. Structures such as leaves, which contain considerable chlorophyll, should be first boiled in slightly alkaline 85% alcohol until the green color is removed.     

Contextual determinants of female-female mounting in laboratory rats

Female-female mounting is widespread among mammalian species, but little is known about the proximal function of this behaviour. While such mounting is often regarded as a 'masculine' trait, its widespread occurrence may indicate that it serves specific functions within the context of female-female social behaviour. We valuated female mounting behaviour in Long-Evans rats in standard observation chambers and in a seminatural enclosure. Under these conditions, we examined a number of potential factors that might influence mounting, including the oestrous cycle, social hierarchy, familiarity and male presence. The female's mounting was not influenced by her own oestrous cycle, but did vary with the oestrous cycle of the stimulus female. Socially dominant females mounted significantly more than subordinate females, and mounting by the dominant female was most frequent when the subordinate female was sexually receptive. Females mounted (and fought with) unfamiliar females significantly more than they did with familiar cagemates. Female-female mounting was dramatically reduced when males were present. Further testing showed that female mounting did not affect the induction of the progestational state of pregnancy, suggesting that female mounting does not function as a pseudomale behaviour that can substitute for genital stimulation provided by the male. Based on these data, female mounting does not appear to function as a sexual behaviour per se, but may serve as a form of female social behaviour related to maintenance of the female's social status within female groups. In this regard, the results of this study suggest that female mounting is part of the normal female's complex behavioural repertoire and does not necessarily reflect masculinization of some underlying neural substrate. Copyright 1999 The Association for the Study of Animal Behaviour.     

Effect of saponin immersion on enhancement of the immune response of white shrimp Litopenaeus vannamei and its resistance against Vibrio alginolyticus

The haemocyte count, phenoloxidase (PO) activity, specific alpha(2)-macroglobulin (alpha2-M) activity, respiratory burst, superoxide dismutase (SOD) activity, glutathione peroxidase (GPx) activity, phagocytic activity, and clearance efficiency against Vibrio alginolyticus were examined when the white shrimp Litopenaeus vannamei (10.42+/-2.0g) were immersed in seawater (34 per thousand) containing different concentrations of saponin (0, 0.5, 1 and 2mgL(-1)) for 24, 48 and 72h. Hyaline cells (HC), the total haemocyte count (THC), specific alpha2-M activity, respiratory burst, SOD activity, and GPx activity directly increased with the saponin concentration, whereas PO activity was inversely related to the saponin concentration. White shrimp L. vannamei that were immersed in saponin at 1 and 2mgL(-1) showed increased phagocytic activity and clearance efficiency to V. alginolyticus over 48-72h. In another experiment, shrimp immersed in seawater containing different concentrations of saponin after 72h were challenged with V. alginolyticus at 3.2x10(5) colony-forming units (cfu)shrimp(-1), and then placed in seawater. The survival rate of shrimp immersed in seawater containing saponin at either dose was significantly higher than that of control shrimp after 12h, as well as at the termination of the experiment (5days after the challenge). It was therefore concluded that L. vannamei immersed in water containing saponin at 2mgL(-1) or less exhibited an immunomodulatory effect, as well as protection against V. alginolyticus infection.     

Endothelium-independent and -dependent contractions induced by endothelin-1 in canine basilar arteries

Endothelium-dependence of contractile responses to endothelin-1 was examined in isolated canine basilar arteries. Within 2 hrs after mounting tissue preparations, endothelin-1 (10(-9) M) caused a monophasic tonic contraction that developed very slowly and was sustained in intact and endothelium-removed arteries. More than 5 hrs after tissue mounting, endothelin-1 (10(-9) M) caused a biphasic contraction consisting of phasic and tonic components in intact arteries, and caused a monophasic tonic contraction in endothelium-removed arteries. This phasic component was significantly decreased by aspirin (5 x 10(-5) M,), OKY-046 (10(-5) M) (a TXA2 synthetase inhibitor) and ONO-3708 (10(-8) M) (a TXA2 antagonist). The present experiments demonstrate that endothelin-1 causes an endothelium-independent tonic contraction and an endothelium-dependent phasic contraction in canine basilar arteries, and suggest that TXA2 plays a role as an endothelium-derived contracting factor.     

Frequent harassment of mounting after a takeover of a group of moor macaques (Macaca maurus)

Harassment behavior during mounting was observed in a group of moor macaques (Macaca maurus) in Sulawesi, Indonesia. During the study periods in 1994 and 1995, most harassment was performed by juveniles less than 5 years old. No harassment by adult females was observed. Mounting by the α-male, who had newly immigrated to the group, was harassed more frequently than that by the β-male in 1994. In 1995, the frequency of harassment during mounting by the α-male was low as compared with that in 1994. In the case of the new α-male, harassment occurred more frequently during mounting with sexually tumescent females than during mounting with detumescent females. Harassment was rarely observed during mounting by natal males of the group. Many hypotheses have been proposed to explain harassment of mounting in primates. The present results offer support for the following two hypotheses: (1) harassment is an attempt to establish a social bond between the performer and the mounting individuals; or (2) harassment occurs in response to mounting by unfamiliar males.     

A method for preparing and staining histological sections containing titanium implants for light microscopy

An improved method for preparing and staining ground tissue-implant sections for light microscopy is presented. Undecalcified tissue blocks with titanium implants were dehydrated in an ascending series of ethanol and stained in toto with basic fuchsin. Specimens were infiltrated and embedded in methyl methacrylate and sections were prepared using a cutting-grinding-system. The polished surface was counterstained with light green or anilin blue. Light polymerizing resin was used as slide mounting medium and for mounting the coverglass. The sections obtained were 10-15 microns thick with tissue architecture which clearly differentiated structures at the tissue-implant interface. The method was very useful for computer assisted morphometric analysis.     

一种用于食草动物粪便显微组织分析的临时装片新技术

粪便显微组织分析法是研究食草动物食性的主要方法,其常规装片技术需要使用Hoyer's 装片介质对植物碎片进行封片,而Hoyer's 封片液的粘性易导致植物碎片在装片过程中发生卷曲和重叠,影响植物碎片的识别效果。本文提出的新装片技术采用没有粘性的饱和NaCl 溶液代替Hoyer's 装片介质,结合特定的定量取样方法和装片程序,可以有效地减少植物碎片的卷曲率和重叠率。对比试验显示,新装片技术可使植物碎片卷曲率从10.4% 下降至3.8%,重叠率从25% 下降至8.1% ,说明新装片技术在减少植物碎片卷曲和重叠方面明显优于常规装片方法。     

Some secrets of fluorescent proteins: distinct bleaching in various mounting fluids and photoactivation of cyan fluorescent proteins at YFP-excitation

Background

The use of spectrally distinct variants of green fluorescent protein (GFP) such as cyan or yellow mutants (CFP and YFP, respectively) is very common in all different fields of life sciences, e.g. for marking specific proteins or cells or to determine protein interactions. In the latter case, the quantum physical phenomenon of fluorescence resonance energy transfer (FRET) is exploited by specific microscopy techniques to visualize proximity of proteins.

Methodology/Principal Findings

When we applied a commonly used FRET microscopy technique - the increase in donor (CFP)-fluorescence after bleaching of acceptor fluorophores (YFP), we obtained good signals in live cells, but very weak signals for the same samples after fixation and mounting in commercial microscopy mounting fluids. This observation could be traced back to much faster bleaching of CFP in these mounting media. Strikingly, the opposite effect of the mounting fluid was observed for YFP and also for other proteins such as Cerulean, TFP or Venus. The changes in photostability of CFP and YFP were not caused by the fixation but directly dependent on the mounting fluid. Furthermore we made the interesting observation that the CFP-fluorescence intensity increases by about 10 - 15% after illumination at the YFP-excitation wavelength – a phenomenon, which was also observed for Cerulean. This photoactivation of cyan fluorescent proteins at the YFP-excitation can cause false-positive signals in the FRET-microscopy technique that is based on bleaching of a yellow FRET acceptor.

Conclusions/Significance

Our results show that photostability of fluorescent proteins differs significantly for various media and that CFP bleaches significantly faster in commercial mounting fluids, while the opposite is observed for YFP and some other proteins. Moreover, we show that the FRET microscopy technique that is based on bleaching of the YFP is prone to artifacts due to photoactivation of cyan fluorescent proteins under these conditions.