Selective recognition and imaging of bacterial model membranes over mammalian ones by using cationic conjugated polyelectrolytes.

The development of new tools for the detection and fluorescence imaging of bacteria is of great interest in clinical diagnosis and food and environmental safety. In this work, we have explored the ability of two cationic fluorene-based conjugated polyelectrolytes, HTMA-PFP and HTMA-PFNT, emitting in the blue and red spectral regions respectively, to selectively label bacterial over mammalian cells. With this end in view, vesicles with lipid compositions mimicking those of bacterial or mammalian membranes were used as model membranes to explore the interaction of the polyelectrolytes with both systems in samples containing either a single type of vesicle or a mixture of both. Changes in the intrinsic fluorescence of HTMA-PFP and HTMA-PFNT were used to quantify the affinity of these polyelectrolytes for the model lipid membranes, while quenching experiments were employed to evaluate their selectivity to each lipid system. In addition, fluorescence microscopy experiments were performed to check the ability of polyelectrolytes to label the vesicles without affecting their integrity. Results showed that both polyelectrolytes rapidly label the model vesicles but they preferentially bind to those mimicking bacterial membranes, HTMA-PFNT being much more selective to this type of membranes than HTMA-PFP. Preliminary experiments with living bacteria and mammalian cells support this conclusion, showing that in samples with both types of cells together, HTMA-PFNT only images the bacterial cells, thus evidencing its potential use for the selective recognition and imaging of bacterial presence.

Cyclooxygenase-2 expression in astrocytes and microglia in human oligodendroglioma and astrocytoma.

Cyclooxygenases (cox) are potent mediators of inflammation and two cox-isoenzymes, cox-1, cox-2, are described to date. Cox-2 is cytokine-inducible in inflammatory cells and enhanced cox-2 expression has been attributed a key role in the development of edema and immunomodulation in pathologically altered brain tissues. In normal cerebral cortex cox-2 is present only in neurons, but not in the glial or vascular endothelial cells. The function of microglia in glioma biology is unclear. Microglia have both neurotrophic and neurotoxic functions and have been shown to release a variety of cytokines. Our preliminary results showed that the expression pattern of cox-2 is predominantly neuronal although glial expression was observed with the correlation of high malignancy. In this study we aimed to assess the phenotypes (astrocyte, microglia) of the cox-2-expressing glial cells in various types of human gliomas and to compare their expression patterns. For this purpose we employed dual immunohistochemistry for cox-2 and GFAP (astrocyte) or LCA-MAC (microglia-macrophage) in archival formalin-fixed, paraffin embedded human tissue diagnosed as oligodendroglioma and/or astrocytoma. The results showed that cox-2 immunoreactivity is up-regulated in the neurons according to the tumor grade. Most of the cox-2 immunoreactive glia were GFAP-positive in anaplastic oligodendrogliomas and at lesser extend in glioblastomas. Cox-2 and LCA co-localization was detected in more glial cells in glioblastomas. It may be speculated that the induction of cox-2 in microglia may contribute to the deleterious effects of prostanoids in cerebral edema formation during the progression of oligodendrogliomas. The detection of cox-2 in astrocytes surrounding the necrotic areas might be important to develop new strategies, such as the usage of cox-2 inhibitors combine with chemotherapy and radiotherapy in the treatment of glioma patients.

Immunohistochemical detection of p53 protein in basal cell skin cancer after microwave-assisted antigen retrieval.

p53 is the most frequently altered tumor-suppressor gene in skin cancer. In normal tissues the p53 protein (wild type) has a very short half-life and it is not detectable immunohistochemically. In contrast, the mutant p53 protein has an extended half-life in tumor cells and can be detected by immunohistochemical methods. p53 is widely used as an indicator of tumor aggression and progression. Fixation methods especially formaldehyde based fixation may mask the immunohistochemical detection of p53 protein but antigen retrieval methods enhance the inmmunohistochemical detection of p53 protein by remodification of protein structure. This study was designed to evaluate the efficacy of different fixatives, of microwaving and microwave pretreatment method to retrieve p53 immunoreactivity in paraffin-embedded non-lesioned (adjacent normal tissue) human skin samples or pathological human skin samples diagnosed as basal cell carcinoma. The samples were fixed at RT and/or in microwave oven either in neutral buffered formalin or alcohol for different time periods. For antigen retrieval, the sections were irradiated in a microwave oven for 5 cycles in 10 mM citrate buffer (pH 6.00). In this study the effects of six different fixation methods on the immunohistochemical staining have been investigated in basal cell tumor specimens. The application of antigen retrieval method was also examined and compared. Optimal results were obtained using samples fixed in alcohol either at room temperature (24 h) or in microwave oven.

Using microwave irradiation in Marchi's method for demonstrating degenerated myelin.

Conventional methods for histological preparation of degenerated myelin are time-consuming and difficult. The purpose of our study was to shorten the time required for the procedure and to obtain better quality results for light microscopic demonstration of degenerated myelin in the central and peripheral nervous systems by using microwave irradiation. Rat brain and sciatic nerve were used for the study. The middle cerebral artery was occluded and the sciatic nerve was cut to produce myelin degeneration. Marchi's method was used for staining degenerated myelin. Fixation for light microscopy that would take two days using the conventional procedure was completed in 16.5-18.5 min using microwave irradiation. While staining of degenerated myelin requires 10 days for the conventional Marchi method, we decreased it to 7 h for brain tissue and 1 h for sciatic nerve by using the microwave oven. Moreover, a better quality preparation was achieved in the groups stained under microwave irradiation than those prepared by the conventional method.

A simple and rapid microwave-assisted hematoxylin and eosin staining method using 1,1,1 trichloroethane as a dewaxing and a clearing agent.

The use and practicability of microwave-assisted staining procedures in routine histopathology has been well established for more than 17 years. In the study reported here, we aimed to examine an alternative approach that would shorten the duration of dewaxing and clearing steps of hematoxylin and eosin (H & E) staining of paraffin sections by using a microwave oven. Although xylene is one of the most popular dewaxing and clearing agents, its flammability restricts its use in a microwave oven; thus we preferred 1,1,1 trichloroethane, which is not flammable, as the dewaxing and clearing agent in the present study. In Group I and Group II (control groups), intestine was processed with xylene and 1,1,1 trichloroethane, respectively. The sections were then stained with H & E according to the conventional staining protocol at room temperature and subdivided into two groups according to the duration of dewaxing and clearing in xylene. In Groups III and IV (experimental groups) similar tissues were processed with xylene and 1,1,1 trichloroethane, respectively; however, sections from these groups were divided into four subgroups to study the period required for dewaxing and clearing in 1,1,1 trichloroethane, then stained with H & E in the microwave oven at 360 W for 30 sec. Our conventional H & E staining procedure, which includes dewaxing, staining and clearing of sections, requires approximately 90 min, while our method using 1,1,1 trichloroethane and microwave heating required only 2 min. Our alternative method for H & E staining not only reduced the procedure time significantly, but also yielded staining quality equal or superior to those stained the conventional way. Our results suggest that 1,1,1 trichloroethane can be used effectively and safely as a dewaxing and clearing agent for H & E staining in a microwave oven.

A comparison of microwave heating and proteolytic pretreatment antigen retrieval techniques in formalin fixed, paraffin embedded tissues.

Antigen retrieval (AR) is a technique that re-exposes epitopes in formalin fixed, paraffin embedded sections and makes them detectable by immunohistochemistry. We compared the effects of two AR procedures, enzyme digestion and microwave heating, on immunostaining of vimentin and desmin in formalin fixed, paraffin embedded tissues. Our results showed that AR is necessary for vimentin and desmin immunostaining in tissues fixed in formalin for more than 48 h. With prolonged fixation times, microwave heating showed better results than enzyme digestion for AR. The same results were obtained using 1% zinc sulfate or Citra Plus solution as retrieval solutions for microwave heating. We recommend microwave heating for AR, because it is easier to use and produces better results compared to enzyme treatment.

Rapid Bielschowsky silver impregnation method using microwave heating.

The Bielschowsky silver impregnation method has been used extensively to demonstrate neuronal processes including dendrites, axons and neurofibrils. In this study, we examined the differences in the time required for and the staining quality of the Bielschowsky method for neuronal processes when microwave heating was used instead of processing at room temperature. For this purpose, a control group of sections stained according to the conventional method at room temperature was compared to an experimental group stained in a microwave oven at 180 W for 2, 4 and 1 min in 2% silver nitrate, ammoniacal silver nitrate and gold chloride, respectively. Light microscopic examination demonstrated that the normal structure was preserved in both groups and that there was no difference in the staining quality between the control and the microwave groups. In addition, staining time for this procedure was reduced to 8 min by using the microwave oven. Our study revealed that microwave irradiation can be used safely for Bielschowsky silver impregnation of neuronal tissues.

Safranin O staining using a microwave oven.

We investigated the effects of microwave irradiation on a safranin O staining method for paraffin sections of formalin fixed rabbit larynx. The control sections were stained according to the conventional method, and the experimental sections were stained in microwave oven for 10 sec at 360 W in Weigert's iron hematoxylin, and for 30 sec at 360 W in fast green and 0.1% safranin O staining solutions. Light microscopic examination of the sections revealed that the microwave heating did not adversely affect the staining properties of cartilage tissue compared to the conventional staining method. Small differences such as darker staining of the matrix and shrinkage of the cytoplasm was observed in some microwave treated sections. The present study revealed that microwave application can be used safely for the safranin O method with the advantage of reduced staining time.

An experimental study of bilateral repositioning of the Stensen's duct orifices with autologous vein and artery grafts in dogs.

Sialorrhoea is an indication of dysfunction in the coordination of the swallowing mechanism. Disturbance in this coordination results in excess pooling of saliva in the anterior mouth and resultant drooling. Several surgical techniques have been described for the management of sialorrhoea. In this experimental study, we planned to try a more safe and physiologic technique by repositioning of the parotid duct orifices into the glossopalatal arch, using autologous free grafts of vein and artery in dogs. Ten adult dogs were used. In each dog, both of the parotid duct orifices were included in the study. The surgical procedure involved the dissection of both parotid duct orifices and their relocation via a submucosal tunnel into the glossopalatal arch, using a vein graft for the right side and an artery graft for the left side. Functional assessment was based on the clinical observations and retrograde sialography done on the 60th day. Results were excellent. No stricture or obstruction was noted. Histological examinations done on the 90th day showed the replacement of endothelium by multilayered cubic Stensen's duct epithelium in both artery and vein grafts. There was no difference between the results of artery and vein grafts. Surgical transposition of Stensen's duct into the glossopalatal arch with autologous vein or artery graft is a safe technique which may be used in clinical cases of drooling as an alternative to the other techniques described.

Effects of microwave irradiation and chemical fixation on the localization of perisinusoidal cells in rat liver by gold impregnation.

Modified gold impregnation is one of the methods that are used in light microscopical demonstration of hepatic perisinusoidal cells. This method has some disadvantages, such as restriction of fixation time to 16 h, which allows limited time for processing the tissues, especially when dealing with a large amount of material, and a long impregnation time (16-24 h). We investigated the effect of prolonged fixation on the staining of sections, to shorten the time needed for gold impregnation by using microwave irradiation. Liver specimens were fixed in Baker's calcium-formalin for different periods of time. After fixation, frozen sections were impregnated in gold chloride solution either at room temperature or in a microwave oven. The staining quality of the sections which had been impregnated in the microwave oven for a much shorter time were equal to or even superior to the ones impregnated at room temperature. Prolonging the fixation time up to 7 days did not affect the staining results by microwave irradiation, whereas satisfactory results were not obtained from sections stained at room temperature and fixed for more than 3 days. We conclude that microwave irradiation can be used to shorten the impregnation time in gold chloride solution and the duration of fixation can be prolonged up to 3 days in the original method and up to 7 days when microwave irradiation is used during impregnation.

Rapid staining of ultrathin sections with the use of a microwave oven.

The microwave oven has many potential applications, ranging from tissue fixation to staining for light and electron microscopy. This study was planned to speed up the staining of ultrathin sections. The first set of grids was stained conventionally with uranyl acetate and Reynold's lead citrate solutions. The other grids were stained with the same solutions by microwave irradiation. The electron micrographs of grids stained using the microwave technique were as satisfactory as the grids stained conventionally. Microwave-treated grids demonstrated more uniform staining and less precipitate. The use of a microwave oven shortened staining time by approximately 38 min.

Microwave fixation of whole fetal specimens.

This study compares microwave fixation of whole fetal specimens with conventional techniques performed at room temperature. All fetuses were obtained from the same pregnant rat; half of them were placed in neutral formalin for 15 min at room temperature, then irradiated for 2.5 min in a domestic microwave oven. The remaining fetuses were placed in neutral formalin at room temperature for 48 hr as a control. Both experimental and control groups were exposed to routine tissue processing for light microscopy and embedded in paraffin wax. Sections 5 microns thick were stained with hematoxylin and eosin. Our results showed that the microwave technique reduced the fixation time while providing thin sections that were equal to or better in quality than those in the control group.

Urethral reconstruction with autologous vein graft: an experimental study.

There is no universally accepted material for urethral reconstruction. This study presents the results of segmental urethral replacement with a free graft of jugular vein in rabbits. Histological examination showed ingrowth of normal transitional epithelium into the venous endothelium. Retrograde urethrograms revealed an excellent result up to 300 days. Fistulas and infection occurred in 4/44 rabbits; these settled spontaneously. No structures or papillary hypertrophy were noted. Segmental urethral reconstruction with autologous vein graft is a simple technique with few complications and appears suitable for use in clinical cases.