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PubMed1987

Pitfalls of immunogold labeling: analysis by light microscopy, transmission electron microscopy, and photoelectron microscopy.

The immunogold method is widely used to localize, identify, and distinguish cellular antigens. There are, however, some pitfalls that can lead to nonspecific binding, particularly in cytoskeletal studies with gold probes prepared from small gold particles. We present a list of suggestions for minimizing nonspecific binding, with particular attention to two problems identified in this study. First, we find that the method used to prepare the colloidal gold particles affects the degree of nonspecific binding. Second, the standard BSA-stabilized small gold probes evidently possess exposed regions that bind to the proteins of cytoskeletal preparations. This was investigated in whole-mount cytoskeletal preparations of cultured cells by use of light microscopy, transmission electron microscopy, and photoelectron microscopy of silver-enhanced specimens. Gold probes were made from approximately 5-nm particles generated by reduction of HAuCl4 with three different reducing agents: white phosphorus, sodium borohydride, and citrate-tannic acid. All three preparations stabilized in the conventional way showed significant levels of nonspecific binding, which was highest with citrate-tannic acid. This problem was largely solved with all three types of probes by including fish gelatin in the probe buffer, by substituting fish gelatin for the BSA stabilizer used to prepare the probes, or by pre-adsorption methods. Application of these techniques resulted in clear immunogold labeling patterns with minimal nonspecific background.

Actins↗
PubMed1982

Substructure of granules from serous cells of porcine tracheal submucosal glands.

The ultrastructure of serous cells from porcine tracheal submucosal glands was studied by conventional transmission electron microscopy (TEM), and by cytochemical methods to stain for complex carbohydrates. In tissue fixed and processed for TEM, and stained with uranyl acetate and lead citrate, the condensing granules of serous cells occasionally possessed a hexagonal and sometimes a lamellar substructure. Tissue fixed in paraformaldehyde-glutaraldehyde and stained with periodic acid-thiocarbohydrazide-silver proteinate (PTS) or with phosphotungstic acid (PTA) showed secretory granules stained for complex carbohydrates and revealed a substructure similar to that noted in the condensing granules. The dark staining substructure revealed by either the PTS or the PTA technique appeared to correspond to electron-lucent areas observed in the condensing granules by conventional TEM. The PTS staining probably demonstrated the presence of neutral glycoprotein, since the serous-cell granules did not react with a dialyzed iron stain for acidic glycoproteins. Treatment of periodic acid oxidized thin sections with pronase or pepsin prior to thiocarbohydrazide and silver proteinate treatment decreased the intensity of the PTS staining, but did not digest away any components of the granules. The substructure revealed by the carbohydrate stains may be a reflection of the mechanism of packaging or the macromolecular structure of the glycoproteins in the serous-cell granules.

Animals↗
PubMed2006

Recovery in a patient with locked-in syndrome.

BACKGROUND AND PURPOSE: Sildenafil citrate has been shown to enhance neurogenesis, angiogenesis, synaptogenesis, and neurological outcome by augmentation of cyclic guanosine monophosphate (cGMP) levels in animal models of ischemic stroke. Whether sildenafil citrate may be helpful for recovery in human stroke is unknown at this time. METHODS: A 41-year-old woman with locked-in syndrome due to pontine infarction began receiving 150 mg of oral sildenafil citrate daily on a compassionate use basis in August 2003 and continues treatment at this time. Magneto-encephalography (MEG) was performed at 12 and 17 months after stroke. RESULTS: No serious adverse events have occurred. Significant milestone recoveries including standing, use of both arms, talking, and full return of swallowing have occurred, particularly after nine months of treatment. The MEG showed a significantly increased amplitude in the somatosensory cortex. CONCLUSION: Daily use of high dose sildenafil citrate appears to be safe in this patient with stroke resulting in locked-in syndrome. Further studies will be required to establish safety and efficacy.

Adult↗
PubMed2003

Opacification of hydrophilic acrylic intraocular lenses: a clinical and ultrastructural analysis of three explanted lenses.

BACKGROUND: The purpose of our study was to report the clinical and ultrastructural results of the late opacification of three Hydroview intraocular lenses (IOLs) explanted from three patients with late postoperative visual acuity drop. PATIENTS AND METHODS: Three different patients without any systemic or ocular associated pathology presented with decreased visual acuity 12 to 24 months after uneventful phacoemulsification and Hydroview IOL implantation. The lenses were explanted and examined by photon and transmission electron microscopy (TEM), as well as through laboratory chemical analysis using bleaching with H 2 O 2, SDS-electrophoresis and silver staining for protein detection. They were cut in small pieces, fixed with osmium tetroxide 1.5 %, dehydrated through ascending grades of ethanol, and embedded in EPON 812. Ultrathin sections were stained with aqueous solutions of uranyl acetate and lead citrate, and they were examined with JEOL 2000 CX TEM. One normal IOL was also prepared in the same way and served as control. RESULTS CONTROL: It was transparent and no material on the surface of semithin and thin sections was detected. Cases 1 and 2: A total gray-whitish opacification of these lenses occurred. An amorphous loose material was present on semithin sections in alternation with normal areas. Evaluation with TEM revealed a pattern of network distribution within the lenses material. The latter was continuous and lightly granulated at the periphery, yet irregularly arranged in the center, more intensely stained, leaving areas of normal appearance. After bleaching, no changes in opacification were seen macroscopically. SDS-electrophoresis and silver staining did not detect proteins. Case 3: This lens had a small opacification close to the periphery. CONCLUSIONS: Late opacification of hydrophilic IOLs is not an uncommon phenomenon after uneventful cataract surgery. Although calcified deposits on the surfaces of such lenses have been reported, this is the first study that describes ultrastructural changes in the IOL material itself.

Aged↗
PubMed1986

Hypersensitive teeth. Part II: Treatment.

If the hydrodynamic theory for pain transmission were accepted, occlusion of the patent dentinal tubules would appear to be essential for treatment efficacy. There are many compounds with seemingly varied chemical forms that have been shown to be effective. Their exact mode of action however, is not clearly defined because well-designed, nonbiased, and controlled comparison studies between agents are lacking. The various toothpastes may have ingredients that actually occlude patent tubules or they may cause secondary desensitization by irrational or abrasive action. In any pain study, the nature of the placebo effect and other psychogenic factors play a significant role. Fluoride preparation with and without iontophoresis has been shown to alter tubule structure and form microprecipitates. The natural desensitization process, although slow, is nature's protection, allowing dentinal sclerosis of secondary dentin formation. Although the resin-adhesive systems, especially the new light-cured dentin bonding agents, appear immediately to be effective, the effect on the pulp remains unknown. Perhaps a combination of iontophoresis with sodium fluoride and light-cured dentin bonding material may yield protection and desensitization at a high level of predictability. With the population trends toward a more geriatric society, further research, knowledge, and understanding of dentinal hypersensitivity is of paramount importance. The expected increase in longevity of the dentition suggests that dentin exposure and sensitivity will increase as a clinical problem. There is a clear time-age relationship involved in gingival recession, erosion and attrition of the teeth, and the need for periodontal surgical therapy. For total comprehensive care, patient comfort is important and should be provided along with sound periodontal health and ideal restorative function.

Adhesives↗
PubMed2001

Modified silver staining of nucleolar organizer regions to improve the accuracy of image analysis.

Silver staining of nucleolar organizer regions (NORs) and their subsequent quantification by image analysis are used increasingly in human pathological specimens and experimental models. Because certain conditions determined by the type of tissue and/or its fixation render AgNOR segmentation for image analysis difficult due to insufficient contrast or nonspecific silver precipitation, we propose three improvements to the original technique to overcome these difficulties. Pretreatment with 7% nitric acid produced very distinct dark brown images of AgNORs on a yellow background. The gradient of background colors allowed easy discrimination of nucleolar, nuclear and cytoplasmic structures. Seven morphometric parameters related to number, size and shape of AgNORs were evaluated quantitatively by image analysis on sections pretreated with nitric acid and on adjacent sections treated with citrate buffer in a wet autoclave according to the most widely accepted method for image analysis of AgNOR. Both methods yielded similar results. A second improvement was achieved by coating the slides with 7% celloidin solution in ethyl alcohol-ether prior to AgNOR staining and acid pretreatment. This coating prevented nonspecific silver deposition on argyrophilic bacteria and other tissue debris in human vaginal smears that could make visualizing AgNOR sites difficult. Finally, placing sections face down on the staining solution prevents the formation of nonspecific silver precipitates. These procedures can be applied together or separately according to the requirements of the material to be evaluated.

Adenocarcinoma↗
PubMed2005

Improved Taxol production by combination of inducing factors in suspension cell culture of Taxus baccata.

To date enormous attempts have been devoted to improve Taxol production exploiting various methodologies from bioprocess engineering to biotechnological and synthetic approaches. We have developed a 2-stage suspension cell culture of Taxus baccata L. using modified B5 medium in order to improve cell growth as well as productivity. After callus induction and cell line selection, B5 medium was supplemented with vanadyl sulfate (0.1 mg/l), silver nitrate (0.3 mg/l) and cobalt chloride (0.25 mg/l) at the first day of stage I culture to maximize cell growth. This medium was further supplemented with sucrose (1%) and ammonium citrate (50 mg/l) on day 10 and sucrose (1%) and phenylalanine (0.1 mM) on day 20 (i.e., biomass growth medium). At stage II (day 25), two different concentrations of several elicitors such as methyl jasmonate (10 or 20 mg/l), salicylic acid (50 or 100 mg/l) and fungal elicitor (25 or 50 mg/l) were added to the biomass growth medium with the aim of improving cellular productivity. For morphological analysis, microscopic inspection was carried out during cultivation. Cell-associated and extracellular amount of Taxol were detected and measured using HPLC methodology. At stage I, overall Taxol amount of biomass growth medium was 13.75 mg/l (i.e., 5.6-fold higher than that of untreated B5 control). At stage II, treated cells with methyl jasmonate (10 mg/l), salicylic acid (100 mg/l) and fungal elicitor (25 mg/l) produced the highest amount of Taxol (39.5 mg/l), which is 16-fold higher than that of untreated B5 control (2.45 mg/l). Microscopic analyses of Taxus cells in suspension cultures showed various positional auto-fluorescence showing direct correlation with Taxol production. Our studies revealed that intervallic supplementation of B5 medium with combination of biomass growth factors at stage I and mixture of elicitors at stage II could significantly increase Taxol production. Thus, we suggest that the exploitation of this methodology may improve the production of Taxol since demands for Taxol pharmaceuticals are increasingly growing and resource paucities have limited its direct harvesting from Taxus trees.

Acetates↗
PubMed1977

Histological and ultrastructural study on the subcommissural organ of Bradypus tridactylus.

The subcommissural organ (SCO) of the Bradypus tridactylus was studied from the histological and ultrastructural point of view. The methods of GOMORI (alum-chromic-hematoxylin-phloxine), AZAN (M. Heidenhain), KLUVER and BARRERA (for nerve cells and myelinated fibers) and silver impregnation according to PALMGREN and CAJAL-DE CASTRO, were used. The material prepared for ultrastructural analize was submitted to the sequence of gluteraldehyde, osmium tetroxide, uranyl acetate and lead citrate. In function of the secretory phenomenon, a systematic study is made of different hystological and ultrastructural characteristics of the SCO and hypendymal rosettes, which were considered in the B. tridactylus as a morphofunctional unit. Fine vascular and nerve networks were observed, by the basal parts of the compact tubule alveolar system (hypendymal rosettes) which composes part of the SCO. Nerve fibers were seen between its cells reaching even up to the lumen, and they may end there in bulbar dilatations which seem to show secretory characteristics at the ultrastructural examination.

Animals↗
PubMed2003

An autometallographic technique for myelin staining in formaldehyde-fixed tissue.

A new autometallographic (AMG) technique for staining myelin in formaldehyde- or paraformaldehyde- (PFA) fixed tissue is presented. The tissue sections were exposed to AMG development without prior treatment with silver salts. The method was examined on PFA-fixed tissue from mouse, rat, pig, and formaldehyde-fixed human autopsy material. Samples from brain, spinal cord, cranial, and spinal nerves were either cut on a vibratome, frozen and cryostat sectioned, or embedded and microtome sectioned, before AMG development and counterstaining. The AMG-myelin technique results in a specific black/dark-brown staining of myelin in all parts of the CNS and PNS. It works on all species examined, independent of the histological preparation techniques applied. The AMG staining is stable, stays unchanged through decades, allows counterstaining, and has previously been used with immunohistochemical techniques. On perfusion-fixed tissue the technique works without further fixation, but the intensity of the AMG-myelin staining is increased by increased postfixation time. Additionally, immersion fixation has to last for days depending on the size of the tissue block in order to obtain proper myelin staining. The most feasible explanation of the chemical events underlying the AMG-myelin technique is that nano-sized clusters of metallic silver are formed in the myelin as a result of chemical bounds with reducing capacity, exposed or created by the formaldehyde molecule. The AMG method is simple to perform and as specific as the conventional osmium and luxol fast blue stainings. The present technique is thus an effective, simple, inexpensive, and quick myelin staining method of formaldehyde- or PFA-fixed tissue.

Animals↗
PubMed2004

AuAg bimetallic nanoparticles: formation, silica-coating and selective etching.

A time-resolved study of the formation of AuAg alloy nanoparticles during boiling of AgNO3 and HAuCl4 in the presence of sodium citrate has been performed by monitoring the UV-visible spectra of the solutions. This study reveals clear differences with respect to the formation of pure Au particles, and suggests that gold and silver nanoparticles nucleate separately, but lattice rearrangement eventually leads to formation of alloy nanoparticles with the expected composition, which was confirmed by high resolution TEM. Additionally, we studied the chemical reaction of the alloy nanoparticles with NH4OH, observing that part of the Ag atoms get oxidized, but reorganization of the crystal lattice during the reaction prevents full transformation into pure gold nanoparticles. This allows the synthesis of silica-coated bimetallic particles with tailored plasmon resonance between ca. 430 and 520 nm, as well as homogeneous incorporation of the coated alloy nanoparticles within silica gels.

Journal Article↗
PubMed1999

The cyclin kinase inhibitor p21WAF1/CIP1 is required for glomerular hypertrophy in experimental diabetic nephropathy.

BACKGROUND: Diabetic nephropathy is characterized by glomerular hypertrophy. We have recently shown that experimental diabetes mellitus is associated with an increase in glomerular expression of the cyclin kinase inhibitor p21WAF1/CIP1 (p21). Furthermore, in vitro glucose-induced mesangial cell hypertrophy is also associated with an up-regulated expression of p21. In this study, we tested the hypothesis that p21 mediates diabetic glomerular hypertrophy in vivo. METHODS: Experimental diabetes mellitus was induced by streptozotocin in mice in which p21 was genetically deleted (p21 -/-) and in wild-type mice (p21 +/+). Kidney biopsies were obtained from diabetic and control (citrate injected) p21 +/+ and p21 -/- mice at day 60. The tissue was used for morphologic studies of glomerular size (measured by computer image-analysis system), glomerular cellularity (cell count), glomerular matrix expansion (silver stain), apoptosis (TUNEL), and expression of transforming growth factor-beta1 (TGF-beta1) by in situ hybridization. RESULTS: The glomerular tuft area increased 11.21% in diabetic p21 +/+ mice at day 60 compared with control (3329.98 +/- 244.05 micrometer(2) vs. 2994. 39 +/- 176.22 micrometer(2), P = 0.03), and the glomerular cell count did not change in diabetic p21 +/+ mice at day 60 compared with the control. These findings are consistent with glomerular hypertrophy. In contrast, the glomerular tuft area did not increase in diabetic p21 -/- mice at day 60 compared with the control (3544.15 +/- 826.49 vs. 3449.15 +/- 109.65, P = 0.82), nor was there an increase in glomerular cell count (41.41 +/- 13.18 vs. 46.95 +/- 3.00, P = 0.43). Diabetic p21 +/+ mice, but not p21 -/- mice, developed an increase in proteinuria at day 60 compared with the control. Tubular cell proliferation, measured by proliferating cell nuclear antigen immunostaining, was increased in both diabetic p21 +/+ (2.1-fold) and p21 -/- (7.61-fold) mice compared with controls. Glomerular cell apoptosis did not increase in diabetic mice. Although glomerular TGF-beta1 mRNA levels increased in both strains of diabetic mice at day 60, the glomerular matrix did not expand. CONCLUSIONS: Hyperglycemia was associated with glomerular hypertrophy in p21 +/+ mice. Despite the increase in TGF-beta1 mRNA, diabetic p21 -/- mice did not develop glomerular hypertrophy, providing evidence that the cyclin kinase inhibitor p21 may be required for diabetic glomerular hypertrophy induced by TGF-beta1. The loss of p21 increases tubular but not glomerular cell proliferation in diabetic nephropathy. The absence of glomerular hypertrophy appears protective of renal function in diabetic mice.

Animals↗
PubMed1991

Cerium as amplifying agent--an improved cerium-perhydroxide-DAB-nickel (Ce/Ce-H2O2-DAB-Ni) method for the visualization of cerium phosphate in resin sections.

A new visualization (Ce/Ce-H2O2-DAB-Ni) procedure for cerium (Ce III) phosphate in semithin and ultrathin plastic sections (Epon 812, Lowicryl K4M, glycol methacrylate) of rat kidney tissues that had been incubated before embedding for the demonstration of phosphatases (alkaline and acid phosphatase, 5(1)-nucleotidase, Mg-dependent ATPase) is described. For this purpose the hydrophobic Epon resin was removed in NaOH-ethanol solution, whereas the hydrophilic Lowicryl and methacrylate sections did not required any etching. The primary reaction product Ce III-phosphate was amplified in a Ce III-citrate solution, subsequently oxidized with H2O2 and then visualized in a H2O2 containing DAB-nickel medium (Ce IV-perhydroxy induced DAB polymerization principle). The method yielded a very clear localization of enzyme activity. The final reaction product (DAB-nickel polymers) in 0.5 - 2.0 microns semithin sections is blue-black; the background staining is completely prevented. An increase of the staining contrast was obtained by posttreatment with OsO4 (osmium black formation). Furthermore, the enzyme reaction product could be demonstrated in 40 nm thick ultrathin sections by silver intensification, which utilized the high argyrophilia of the polymerized DAB-nickel complexes. This procedure replaces the earlier published technique.

3,3'-Diaminobenzidine↗
PubMed1984

A quick method for screening platelet dysfunctions using the whole blood lumi-aggregometer.

Patients who present with a clinical history suggesting a bleeding disorder are often tested initially for a clotting defect rather than for platelet dysfunction, due to the length of time necessary to complete a platelet function study in platelet-rich plasma. We have developed a sensitive method for measuring platelet aggregation and release of ATP employing the Whole Blood Lumi -Aggregometer. This method makes it possible to quickly detect patients who require further study for possible platelet function disorders such as cyclooxygenase deficiency, storage-pool defect, thrombasthenia, and von Willebrand's disease. The results obtained with this electrical impedance instrument do not differ from those obtained with the conventional optical method. However, it is now possible to recognize a platelet function defect within 30 min of obtaining a 5 ml sample of citrated whole blood. Further, platelets of unusual size or density are not lost to testing through centrifugation.

Adenosine Triphosphate↗