Ruthenium Red Stain Able Surface Layer on Lung Alveolar Cells; Electron Microscopic Interpretation

Luft's ruthenium red (RR) method was applied to lung tissue. Small blocks of mouse lung were fixed for 1 hr with 1.2% glutaraldehyde at 0-4 C, buffered with 0.067 M cacodylate, pH 7.3 and containing RR, 1 mg/ml. Following fixation, lung blocks were immersed in 0.15 M cacodylate for 10 min and postfixed for 3 hr at room temperature with 2% OsO₄ buffered with 0.067 M cacodylate, pH 7.3, and containing RR, 1 mg/ml. Blocks were dehydrated with ethanol, embedded in Araldite, and ultrathin sections treated with uranyl acetate and lead citrate solutions to enhance contrast of cell structures. Electron micrographs revealed an electron-dense layer coating the exposed surfaces of alveolar cells. This layer corresponded in location and appearance to that observed by other investigators who used colloidal iron techniques.     

Postembedding adjuvants in routine microtomy of paraffin-processed tissues.

Soaking the exposed face of routine paraffin-processed tissue blocks from human autopsies in solutions containing either 60% ethyl alcohol-glycerol 9:1; glycerol-aniline 9:1; 5% aqueous phenol; 5% aqueous Photo-flo, or 10% aqueous ammonium hydroxide for 2--4 hours at 0--4 C resulted in greater ease of ribboning and overall improvement of slide quality in the majority of sections compared to the results with the same blocks soaked only in water at 0--4 C. The use of postembedding adjuvants, especially 10% aqueous ammonium hydroxide, presents a simple, convenient, inexpensive means of increasing section quality with routine as well as refractory paraffin-embedded tissue without sacrificing tissue adhesion, staining qualities or cellular detail.     

Postembedding Adjuvants In Routine Microtomy Of Paraffin-Processed Tissues

Soaking the exposed face of routine paraffin-processed tissue blocks from human autopsies in solutions containing either 60% ethyl alcohol-glycerol 9:1; glycerol-aniline 9:1; 5% aqueous phenol; 5% aqueous Photo-Flo, or 10% aqueous ammonium hydroxide for 2-4 hours at 0-4 C resulted in greater ease of ribboning and overall improvement of slide quality in the majority of sections compared to the results with the same blocks soaked only in water at 0-4 C. The use of postembedding adjuvants, especially 10% aqueous ammonium hydroxide, presents a simple, convenient, inexpensive means of increasing section quality with routine as well as refractory paraffin-embedded tissue without sacrificing tissue adhesion, staining qualities or cellular detail.     

快速打破结缕草种子休眠方法的比较

用水和30%NaOH对结缕草(Zoysia japonica Steud.)种子进行浸种处理,筛选能快速打破结缕草种子休眠的方法.结果表明:用水浸种6d,8d内发芽率仅为14.70%;用30%NaOH浸种120min,8d内发芽率也仅达28.00%;而用水浸泡2d后再用30%NaOH处理40min,8d内结缕草种子的发芽率达到了82.00%.说明用水和30%NaOH综合处理的方法可以快速有效打破结缕草种子休眠,缩短发芽周期,达到快速出苗的目的.     

阿司匹林和复方新诺明对大豆种子萌发及活力的影响

 以大豆(Glycine max)种子为材料,浓度的阿斯匹林和复方新诺明水溶液,在(20±1)℃条件下分种24h和12h, 测定大豆种子萌发过程中的各项生理生化指标以及萌发后的各形态指标实验结果表明：阿斯匹林和复方新诺明水溶液浸种都能有效地提高大豆种子的发芽率和活力指数,还能促进大豆根系的生长。其中以62.5mg·L-1的阿斯匹林和0.5mg·L-1的复方新诺明处理后的效果最为显著。阿斯匹林浸种能使大豆萌芽的相对电导率显著降低,使活性氧清除酶的活性有所提高。复方新诺明浸种则使大豆萌芽的蛋白质含量以及氨基酸总量都有显著的提高。     

A loss in the plasma membrane ATPase activity and its recovery coincides with incipient freeze-thaw injury and postthaw recovery in onion bulb scale tissue

Plasma membrane ATPase has been proposed to be functionally altered during early stages of injury caused by a freeze-thaw stress. Complete recovery from freezing injury in onion cells during the postthaw period provided evidence in support of this proposal. During recovery, a simultaneous decrease in ion leakage and disappearance of water soaking (symptoms of freeze-thaw injury) has been noted. Since reabsorption of ions during recovery must be an active process, recovery of plasma membrane ATPase (active transport system) functions has been implicated. In the present study, onion (Allium cepa L. cv Downing Yellow Globe) bulbs were subjected to a freeze-thaw stress which resulted in a reversible (recoverable) injury. Plasma membrane ATPase activity in the microsomes (isolated from the bulb scales) and ion leakage rate (efflux/hour) from the same scale tissue were measured immediately following thawing and after complete recovery. In injured tissue (30-40% water soaking), plasma membrane ATPase activity was reduced by about 30% and this was paralleled by about 25% higher ion leakage rate. As water soaking disappeared during recovery, the plasma membrane ATPase activity and the ion leakage rate returned to about the same level as the respective controls. Treatment of freeze-thaw injured tissue with vanadate, a specific inhibitor of plasma membrane ATPase, during postthaw prevented the recovery process. These results indicate that recovery of freeze-injured tissue depends on the functional activity of plasma membrane ATPase.     

Block staining of mammalian tissues with hematoxylin and eosin

Various mammalian tissues were stained en bloc with hematoxylin and eosin after fixation and prior to embedding in paraffin wax and sectioning. The choice of fixative is important and best results are obtained using Worcester's Fluid, a combination of saturated aqueous mercuric chloride, formaldehyde, and glacial acetic acid. After fixation, blocks of tissue up to 1.5 cm thick are stained for seven days in hematoxylin. Excess stain is removed by washing tissues in running water overnight. Tissue blocks then are dehydrated with graded concentrations of ethyl alcohols to 80% and counterstained, with further dehydration, in 0.5% spirit soluble eosin in 90% ethyl alcohol for five days. The tissue is subsequently transferred to 90% ethyl alcohol overnight to differentiate eosin staining; dehydration is completed in absolute ethyl alcohol. The blocks are cleared in in cedarwood oil and briefly in xylene prior to embedding, sectioning, and mounting. Following removal of wax by xylene, coverslips are applied. General morphological and histological features were particularly well differentiated and very selectively and reliably stained by this method.     

Block Staining of Mammalian Tissues with Hematoxylin and Eosin

Various mammalian tissues were stained en bloc with hematoxylin and eosin after fixation and prior to embedding in paraffin wax and sectioning. The choice of fixative is important and best results are obtained using Worcester's Fluid, a combination of saturated aqueous mercuric chloride, formaldehyde, and glacial acetic acid. After fixation, blocks of tissue up to 1.5 cm thick are stained for seven days in hematoxylin. Excess stain is removed by washing tissues in running water overnight. Tissue blocks then are dehydrated with graded concentrations of ethyl alcohols to 80% and counterstained, with further dehydration, in 0.5% spirit soluble eosin in 90% ethyl alcohol for five days. The tissue is subsequently transferred to 90% ethyl alcohol overnight to differentiate eosin staining; dehydration is completed in absolute ethyl alcohol. The blocks are cleared in cedarwood oil and briefly in xylene prior to embedding, sectioning, and mounting. Following removal of wax by xylene, coverslips are applied.

General morphological and histological features were particularly well differentiated and very selectively and reliably stained by this method.     

Antigen retrieval by heating en bloc for pre-fixed frozen material.

Antigen retrieval (AR) is frequently required for successful immunohistochemistry (IHC) in archival formalin-fixed, paraffin-embedded tissue sections. Although AR by heating is most generally used, the majority of existing methods are useful only for paraffin-embedded sections. This article describes a simple alternative method for AR that can be used for aldehyde-fixed frozen sections. After fixation in paraformaldehyde, tissue blocks were heated in retrieval solutions and then frozen with dry ice. The optimal temperatures for heating were 90C and above, and the optimal retrieval solutions were distilled water and 10 mM sodium citrate, pH 6.0. Sections were cut with a cryostat and mounted on poly-l-lysine-coated glass slides. After the sections dried, routine IHC was performed. Alternatively, free-floating sections were used. This method not only greatly enhanced the immunoreactivity for a wide range of antigens, especially for nuclear proteins, but also effectively lowered the background staining in some cases. I examined the staining of 14 antibodies using sections of mouse brain and rat testis. The heating process was essential for five antibodies, improved immunoreactivity for seven antibodies, and provided no change for two antibodies.     

Rapid microwave fixation--a comparative morphometric study

Summary Tissue blocks taken from healthy human lung tisues, from primary bronchial carcinoma and from mediastinal and hilar lymph nodes were placed in the following solutions. Tris buffer; buffered formalin (0.5%, 1%, 7%); 0.1 mol NaCl; distilled water; DMSO (1%, 10%, 20%); acetone (10%); methanol (50%, 80%, 100%); glutaraldehyde (2.5%), and fixed by use of a commercial microwave oven.Tissue blocks obtained from the same surgical specimens were fixed in 7% buffered formalin for 24 h for comparison. Conventional and microwave-fixed tissue was embedded in paraffin, and stained with haematoxylin and eosin. Fifteen specimens of each group and each solution were examined by light microscopy. Minimum diameter and area of 100 nuclei of each specimen were measured interactively.Histomorphological sections fixed with Tris buffer in a microwave oven revealed best morphological results showing more contrast in chromatin distribution of nuclei and opening of interstitial lung capillaries in comparison to conventional formalin-fixed specimens. No statistically significant differences in area and minimum diameter of nuclei between the different groups were found. Microwave fixation using Tris buffer is a time-saving fixation method at least comparable to conventional formalin fixation. It is not accompanied by hazards to the environment that are unavoidable by formalin fixation.     

Glutaraldehyde Inactivation of Virus in Tissue

High concentrations of influenza virus and T3 coliphage were inoculated into mouse tissue blocks. Exposure of the inoculated tissue blocks to 5% alkaline glutaraldehyde resulted in rapid inactivation of both viral agents.     

Adsorption of mutagens to cotton bearing covalently bound trisulfo-copper-phthalocyanine

A method for separating nonpolar mutagens from their dilute aqueous solutions is described. It utilizes the affinity of the mutagens to a phthalocyanine derivative attached to cotton through a covalent bond. For mutagens having 3 or more fused aromatic rings in their structures, efficient adsorption took place on soaking the cotton in their solutions. The mutagens adsorbed can be recovered by elution with ammoniacal methanol. Mutagenicity in smoker's urine, cooked beef, and river water was detected by use of this method.     

Diffusion of KCl in an amylose film

A method has been developed measuring the diffusion coefficient of KCl in amylose films. The films were soaked in potassium chloride solutions, then immersed in pure water and conductivity measured as a function of time. Different concentrations of the soaking solution were used and the measurements were made at several temperatures. The diffusion coefficient of KCl was found to be independent of the soaking solution KCl concentration, but found to increase with increasing temperature. The diffusion coefficient values were about one quarter of those found in water and varied from 4.8×10⁻¹⁰ to 11×10⁻¹⁰ m² s⁻¹. The activation energy of diffusion was close to that found in water. Two values for the activation energy were obtained, 20.1 and 14 kJ mol⁻¹, indicating a change in the film structure at 45 °C. Amylose films swelled equally in KCl-solutions and water. The thickness of amylose films doubled and the increase in mass was 100–200% corresponding the decrease of amylose content from about 87 to 37%, when the conditions changed from normal humidity conditions to water.     

Banking of fresh-frozen prostate tissue using the alternate mirror image protocol: methods, validation, and impact on the pathological prognostic parameters in radical prostatectomy

We evaluated the value of the ‘alternative slices mirror image method’ used in prostate tissue banking in terms of predicting the sampling of cancerous tissue while preserving the pathological prognostic information. The concordance of diagnosis between banked sections and their mirror image paraffin- sections was studied using 50 cases corresponding to 400 H&E sections taken from 400 banked frozen blocks (two presumed benign and two presumed cancer for each case). The mean number of paraffin blocks in each case was 21. On average 29% of the prostate gland was banked and banked tissue contained cancer in 47 cases (94%). There was no difference between the concordant and discordant groups in terms of the final Gleason score, pathological stage, prostate size, number of banked blocks and the percentage of the prostate submitted for banking. However, concordant cases had larger foci of cancer in the mirror image paraffin block (P?=?0.0088). In addition, the surgical margins sections which are not banked using this method provided important information about the pathological stage, surgical margins status and the final Gleason score in 2.6, 2.6, and 1.3% of cases, respectively. The ‘alternative slices mirror image method’ is a straightforward method that is highly efficient in banking prostatic cancerous tissue. Overall, tumor volume and especially size of tumor foci in the image paraffin block are the most important factors in dictating the success rate of banking frozen cancerous tissue. Including ‘surgical margins’ sections for histology provides additional important prognostic information in a minority of cases.     

Silver Staining of Nerve Fibers in Cardiac Tissue

Fresh hearts of dog were perfused through the coronary vessels with 1000 ml. of fixative (chloral hydrate, 5 g. per 100 ml. of 70% ethyl alcohol) and blocks of tissue 2 × 5 mm. from epicardium to endocardium fixed 48 hours in the same fixative. The blocks were placed in 95% alcohol containing 0.3% addition of strong ammonia for 4 hours, followed by 2 changes of plain 95% alcohol of 1 hour each, then cleared and infiltrated with paraffin. Mounted sections 12-15 µ thick were incubated in 1% silver proteinate (obtained from Serumvertrieb, Marburg, Germany)² at 38° C. for 48 hours in the presence of 10 g. of 15 gauge copper wire per 200 ml. of solution. The slides were rinsed gently in 3 changes of distilled water for 2 minutes, 1 minute and 1 minute, respectively, and reduced in 1% hydroquinone and 5% sodium sulfite for 5 minutes. They were washed 5 minutes in tap water and 5 minutes in 2 changes of distilled water and toned 3-5 minutes in 0.25% gold chloride, rinsed in distilled water 10 seconds, reduced 10 seconds in 1 % oxalic acid, rinsed 1 minute, fixed in 5% sodium thiosulfate 5 minutes, washed in tap water through 3 changes, dehydrated, cleared and covered. All solutions were made with distilled water except where otherwise specified. The results gave good impregnation of fine nerve fibers without the usual confusing staining of reticular tissue.     

Nano silver treatment is effective in reducing bacterial contaminations of Araucaria excelsa R. Br. var. glauca explants

The downside of plant tissue culture techniques is an unwanted microbial contamination. Elimination of contaminants is the first step of any successful investigation on plant tissue culture. Preliminary experiments on Araucaria excelsa R. Br. var. glauca (Norfolk-Island pine) (syn.: A. heterophylla) showed that most common decontaminants could not successfully eliminate the contamination. Therefore, nano silver (NS) colloids were evaluated for controlling contamination. Treatments were included soaking the explants in NS solution or adding NS to the culture medium. Explants were cultured on MS medium supplemented with appropriate growth regulators for their establishment. Results showed that surface sterilization followed by treatment with 200 mg l-1 of NS with soaking time of 180 min reduced the bacterial contamination from 61.5% to 11.3% and adding 400 mg l-1 NS to the medium reduced the bacterial contamination from 81.25% to 18.75%. Nano silver could be applied without adverse effects on plant growth and development. This is the first report on in vitro establishment of A. excelsa R. Br. using NS to reduce bacterial infections.     

Auxin transport in explants of coleus

α-Naphthaleneacetic acid-C¹⁴, labeled in the carboxyl group, was applied in blocks of agar to the distal and to the proximal (either apical or basal) ends of explants of Coleus. The radioactivity in receiver blocks at the opposite ends was measured. Acropetal transport was slight, only 4% of the basipetal transport.

Translocation of NAA-C¹⁴ was polar in basipetal direction. Only 1.4% of the radioactivity lost from donor blocks at the apical position reached the receiver blocks; the greatest part remained in the tissue and was immobilized there. All activity found in receiver blocks at the basal end appeared to be still in the form of NAA. There were no differences between petiole tissue and stem tissue, so far as the transport of NAA is concerned.

     

In vitro sodium fluoride exposure decreases torsional and bending strength and increases ductility of mouse femora

Fluoride exposure in vivo can reduce the material strength of bone, an effect that has been attributed to a change in mineral structure. An in vitro model of fluoride exposure offers the potential to study directly the effects of fluoride on bone mineral. Previous investigators have reported that soaking bones in sodium fluoride in vitro reduces bone strength. However, long soaking times and the absence of physiological buffering ions from their treatment solutions may have caused mineral dissolution that contributed to the decrease in bone strength. Our objectives were to further characterize the effects of in vitro fluoride exposure on bone mechanical properties and to determine if the changes reported in previous studies of bovine cortical bone would be observed for whole rodent bones. We soaked 60 mouse femora in sodium fluoride solutions, with and without physiological buffering ions, and evaluated their torsional and bending properties. Fluoride soaked bones had a 30-fold increase in fluoride content and a 23% increase in water content compared to controls. These changes were associated with average reductions in ultimate load of 45%, reductions in rigidity of 70%, and increases in deformation to failure of 80%. The effect of fluoride was similar for bones treated in buffered and non-buffered solutions, and was observed in both torsion and bending. Our findings confirm those of previous studies and highlight the strong effect that in vitro fluoride exposure has on bone mechanical properties. The in vitro model of fluoride exposure offers a tool to further study the effects of ion substitution in bone.     

Role of proline and glycinebetaine pretreatments in improving heat tolerance of sprouting sugarcane (<Emphasis Type="Italic">Saccharum</Emphasis> sp.) buds

High temperature strongly hampers the plant growth particularly at early growth stages. In this study, changes in some physiological and anatomical characteristics and possibility of mitigating the adversities of heat stress by soaking sugarcane nodal buds in 20 mM proline and glycinebetaine (GB) solutions have been explored. Heat stress reduced the rate of bud sprouting nonetheless soaking the setts in proline followed by GB was beneficial. In addition, heat stress reduced the bud fresh and dry weights, generated H₂O₂, reduced the tissue levels of K⁺ and Ca²⁺, while increased the osmolytes synthesis in a time course manner. Heat stress also delayed the emergence and expansion of new bud leaves, by restricting the number and area of mesophyll cells. It also caused poor and aberrant development and diffused appearance of mesophyll cells and vascular bundles in the bud leaves. However, soaking of buds in proline and GB solutions substantially reduced the H₂O₂ production, improved the accumulation of soluble sugars and protected the developing tissues from heat stress effects; although proline was more effective than GB. Correlations of various attributes indicated that soaking in GB and proline restricted the H₂O₂ generation, improved K⁺ and Ca²⁺ contents, and increased the concentrations of free proline, GB and soluble sugars eventually improving the heat tolerance of buds. Cost-benefit analysis showed that, considering increase in sprouting of buds, soaking in 20 mM solution of both osmoprotectants is economical.     

Construction of a tissue microarray with two millimeters cores of endometrioid endometrial cancer: factors affecting the quality of the recipient block

The tissue microarray (TMA) method currently is not used to render a primary diagnosis of cancer, but its scientific value has been proved in studies of various cancer types. TMA technology still is not used often for uterine tumors, however. We investigated the repeatability of histological diagnosis of endometrioid endometrial cancer (EEC) using conventional histology and TMA using 2 mm cores. We examined EEC tissues from 171 patients. Formalin fixed, paraffin embedded tissue donor blocks from EEC specimens were selected and examined histologically. Duplicate 2 mm tissue cores were inserted into a TMA recipient block. EEC tissues were examined as hematoxylin-eosin stained sections from the TMAs. EEC tissue was identified in the TMAs in 158 cases (92.4%) and not found in 13 cases (7.6%). On the TMA slides, both EEC positive cores were identified in 129 cases (75.4%), but only one core in 29 cases (17.0%). Among 342 biopsies of the donor blocks (each case in duplicate), EEC was found in 287 cases (83.9%) using the TMA: 124/146 (84.9%) with superficial infiltration, 153/178 (86.0%) with deep myometrial infiltration, and 10/18 (55.6%) without myometrial infiltration. We concluded that two 2 mm tissue cores from a biopsy of a donor block inserted into a TMA recipient block were sufficient to diagnose EEC in more than 90% of cases. EEC was identified in the TMAs with similar frequency with respect to superficial and deep myometrial infiltration. Cases without myometrial infiltration were identified less often.